反义抑制环阿乔醇合成酶基因对人参发根皂苷合成的影响
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摘要
人参皂苷是人参的主要药理活性物质。研究表明,人参皂苷的前体为2,3-氧化角鲨烯,人参中2,3-氧化角鲨烯分别流向人参皂苷和固醇两条支路。环阿乔醇合成酶(CS)和达玛烯二醇合成酶(DS)分别是控制2,3-氧化角鲨烯流向植物固醇和人参皂苷合成途径的关键酶。环阿乔醇合成酶催化2,3-氧化角鲨烯生成环阿乔醇,环阿乔醇经一系列生化反应生成植物固醇。达玛烯二醇合成酶催化2,3-氧化角鲨烯生成达玛烯二醇,达玛烯二醇经一系列生化反应生成人参皂苷。本研究通过反义RNA技术抑制了人参发根CS的表达,使代谢流主要流向人参皂苷,从而达到提高人参皂苷含量的目的。
本文首先利用RT-PCR方法从人参总RNA中扩增出901bp的CS基因片段,并把此片段插入到pMD18-T载体上,进而克隆到大肠杆菌JM109中。然后,把CS基因片段以反义方向亚克隆到植物表达载体pBI121中,并转化发根农杆菌A4。利用阳性发根农杆菌A4侵染3年生的人参根片,获得表达反义CS的人参发根。
Northern blot分析表明,反义CS发根能表达大量的反义CS-RNA,其正义CS-RNA水平降低。同时,反义CS人参发根的固醇合成水平降低,而人参皂苷含量提高。进一步深入研究表明反义CS人参发根的CS酶活性有所降低,而DS酶活性得到提高。此外,反义发根系早期的2,3-氧化角鲨烯水平也有所提高。
Panax ginseng has been used as tonics for several thousand years in China. Previous studies have suggested that the main effective components of ginseng are ginsenosides, which possess many pharmacological activities including anti-cancer, anti-fatigue, anti-aging, and anti-diabetic. Up to now, ginsenosides are mainly produced from cultured ginseng. Ginseng is a perennial plant, sensitive to soil conditions and grows slowly. In our country, the fields for ginseng culture are mainly obtained through cutting forest, while the fields which have been used for ginseng culture can not be reused in 20-30 years, resulting in gradually-reduced fields available for ginseng culture and gradually-decreased economic returns of ginseng culture. Since 1980’s, scientists from several countries have tried to produce ginsenosides by biotechnologies including ginseng cell culture and callus culture; however, productivity obtained by these means is low due to low growth rate. Ginseng hairy roots, which are induced by Agrobacterium rhizogenes and characterized as fast hormone-independent growth, genetic and biosynthetc stability, therefore are thought as the most potential alternative for ginsenoside production. However, compared with ginseng cultured in fields, the ginsenoside contents of ginseng hairy roots are relatively low. This is one of the main bottle-necks for industrialization of ginseng hairy roots. It is an available strategy to improve ginsenoside biosynthesis of ginseng hairy roots by metabolic engineering based on ginsenoside biosynthetic pathway.
Previous studies have suggested that ginsenosides and phytosterols share the same precursor, 2,3-oxidosqualene. The enzymes cycloartenol synthase (CS) and dammarenediol synthase (DS) are the key enzymes responsible for ginsenoside and phytosterol biosynthesis from 2,3-oxidosqualene, respectively. CS catalyzes the transformation from 2,3-oxidosqualene to cycloartenol, which is further transformed into phytosterols through serial biochemical reactions. Under the catalysis of DS, 2,3-oxidosqualene is transformed into dammarenediol, which provides primary molecular skeleton for ginsenosides biosynthesis.
In this study, suppression of phytosterol biosynthesis in P. ginseng hairy roots, which was achieved by antisense-RNA suppression of CS, led to more precursors available for ginsenosides biosynthesis, therefore resulted in enhanced ginsenoside levels. The main contents and conclusions of this paper are as follows.
A 901bp CS gene fragment was amplified through RT-PCR with primers designed based on the P. ginseng CS sequence in GeneBank. The 901bp fragment was cloned into a vector pMD18-T, and transformed into Escherichia coli JM109. Then the target fragment was subcloned into a plant expression vector pBI121 containing a selection marker of kanamycin resistance, in the antisense orientation. The pBI121 vector harboring antisense-CS construct was introduced into Agrobacterium rhizogenes A4 by a conventional method. Antisense-CS ginseng hairy roots were obtained by infecting the root discs of 3-year old P. ginseng with A. rhizogenes containing antisense-CS vector. The positive hairy root lines were screened on MS medium with kanamycin. Northern blot analyses were performed with 3 randomly-selected control hairy root lines (hairy root lines with empty pBI121 vector: C-2, C-4, C-7) and 5 antisense-CS hairy root lines (A-16, A-25, A-28, A-33, A-38). The results indicated that the antisense-CS hairy root lines can transcribe abundant antisense CS-RNA, while there were almost no antisense CS-RNA in control lines. Furthermore the sense CS-RNA levels of antisense lines were remarkably lower than those of control lines.
According to our observations, all antisense-CS lines and control lines presented the typical traits of hairy roots and had no morphological difference. This suggested that antisense suppression of CS had no effect on morphous of ginseng hairy roots. However, in liquid MS medium, the antisense lines exhibited a slower growth than control lines at early stage; but there was no obvious difference in the final biomass. This implied that antisense-CS manipulation had deleterious effects on hairy root growth during early days of the culture cycle; therefore it is persuasive that phytosterols play an important role in ginseng hairy roots growth during early stage of the culture cycle.
Moreover, total phytosterol, 2,3-oxidosqualene and total ginsenoside levels of antisense-CS hairy root lines and control lines cultured in MS liquid medium, were analyzed and compared at appropriate time points of the culture cycle. Total ginsenoside levels in all lines increased rapidly after day 15 and reached a plateau after day 25, suggesting that ginsenosides are primarily synthesized during the later stage of the culture cycle. Before day 15, total ginsenoside levels were comparable in antisense-CS lines and control lines. However, after day 20, except A-33 line, all antisense-CS lines exhibited higher total ginsenoside levels than control lines. It is noteworthy that the total ginsenoside levels were different among antisense-CS lines, of which the A-16 line had the highest total ginsenoside level (about 200% of that of control lines); however there was no obvious difference in total ginsenoside levels of control lines. The total phytosterol levels of antisense-CS lines were lower than those of control lines, implying that phytosterol biosynthesis was suppressed by antisense suppression of CS. In addition, the total phytosterol levels of all lines were relatively stable all through the culture cycle. The 2,3-oxidosqualene levels of control lines were stable during the culture cycle, while the 2,3-oxidosqualene levels of antisense lines were obviously higher at early stage than at the later stage of the culture cycle. The 2,3-oxidosqualene levels of antisense lines were higher than those of control lines at early stage of the culture cycle, but comparable at the later stage.
To further dissect the mechanism of the enhanced ginsenoside levels in antisense-CS lines, DS and CS enzyme activities in antisense and control lines were determined at day 20 of the culture cycle. The results indicated that all antisense lines, except A-33, exhibited higher DS enzyme activities than control lines, while the CS enzyme activities of antisense lines were lower than control lines. As DS and CS are the key enzymes for phytosterol and ginsenoside biosynthesis, respectively; the decrease of CS enzyme activity and increase of DS enzyme activity in antisense lines could primarily account for the decreased phytosterol levels and enhanced ginsenoside levels in antisense lines. The decreased CS enzyme activities in antisense lines should be caused by antisense suppression of CS; while the increased DS activities in antisense lines were possibly induced by the higher 2,3-oxidosqualene level at early stage during the culture cycle.
We can conclude from above analysis that antisense suppression of CS in P. ginseng hairy roots can lead to decreased CS enzyme activity, thus inhibite the biosynthesis of cycloartenol, from which phytosterols derive, and further suppress phytosterol biosynthesis from 2,3-oxidosqualene. As phytosterols and ginsenosides share the same precursor, 2,3-oxidosqualene, suppression of phytosterol synthesis led to more precursor available for ginsenoside biosynthesis, therefore resulted in enhanced ginsenoside levels in antisense-CS P. ginseng hairy roots. Additionally, antisense suppression of CS also led to higher enzyme activity of DS, the key enzyme for ginsenoside biosynthesis. This might be caused by the higher levels of 2,3-oxidosqualene, which is the substrate of DS, during the early stage of the culture cycle of ginseng hairy roots. Antisense suppression of CS had no effects on the morphous of P. ginseng hairy roots, while led to a slow growth rate of P. ginseng hairy roots during early stage of the culture cycle. This study is significant for further investigation of ginsenoside biosynthesis pathway and ginsenoside biosynthesis regulation.
本文首先利用RT-PCR方法从人参总RNA中扩增出901bp的CS基因片段,并把此片段插入到pMD18-T载体上,进而克隆到大肠杆菌JM109中。然后,把CS基因片段以反义方向亚克隆到植物表达载体pBI121中,并转化发根农杆菌A4。利用阳性发根农杆菌A4侵染3年生的人参根片,获得表达反义CS的人参发根。
Northern blot分析表明,反义CS发根能表达大量的反义CS-RNA,其正义CS-RNA水平降低。同时,反义CS人参发根的固醇合成水平降低,而人参皂苷含量提高。进一步深入研究表明反义CS人参发根的CS酶活性有所降低,而DS酶活性得到提高。此外,反义发根系早期的2,3-氧化角鲨烯水平也有所提高。
Panax ginseng has been used as tonics for several thousand years in China. Previous studies have suggested that the main effective components of ginseng are ginsenosides, which possess many pharmacological activities including anti-cancer, anti-fatigue, anti-aging, and anti-diabetic. Up to now, ginsenosides are mainly produced from cultured ginseng. Ginseng is a perennial plant, sensitive to soil conditions and grows slowly. In our country, the fields for ginseng culture are mainly obtained through cutting forest, while the fields which have been used for ginseng culture can not be reused in 20-30 years, resulting in gradually-reduced fields available for ginseng culture and gradually-decreased economic returns of ginseng culture. Since 1980’s, scientists from several countries have tried to produce ginsenosides by biotechnologies including ginseng cell culture and callus culture; however, productivity obtained by these means is low due to low growth rate. Ginseng hairy roots, which are induced by Agrobacterium rhizogenes and characterized as fast hormone-independent growth, genetic and biosynthetc stability, therefore are thought as the most potential alternative for ginsenoside production. However, compared with ginseng cultured in fields, the ginsenoside contents of ginseng hairy roots are relatively low. This is one of the main bottle-necks for industrialization of ginseng hairy roots. It is an available strategy to improve ginsenoside biosynthesis of ginseng hairy roots by metabolic engineering based on ginsenoside biosynthetic pathway.
Previous studies have suggested that ginsenosides and phytosterols share the same precursor, 2,3-oxidosqualene. The enzymes cycloartenol synthase (CS) and dammarenediol synthase (DS) are the key enzymes responsible for ginsenoside and phytosterol biosynthesis from 2,3-oxidosqualene, respectively. CS catalyzes the transformation from 2,3-oxidosqualene to cycloartenol, which is further transformed into phytosterols through serial biochemical reactions. Under the catalysis of DS, 2,3-oxidosqualene is transformed into dammarenediol, which provides primary molecular skeleton for ginsenosides biosynthesis.
In this study, suppression of phytosterol biosynthesis in P. ginseng hairy roots, which was achieved by antisense-RNA suppression of CS, led to more precursors available for ginsenosides biosynthesis, therefore resulted in enhanced ginsenoside levels. The main contents and conclusions of this paper are as follows.
A 901bp CS gene fragment was amplified through RT-PCR with primers designed based on the P. ginseng CS sequence in GeneBank. The 901bp fragment was cloned into a vector pMD18-T, and transformed into Escherichia coli JM109. Then the target fragment was subcloned into a plant expression vector pBI121 containing a selection marker of kanamycin resistance, in the antisense orientation. The pBI121 vector harboring antisense-CS construct was introduced into Agrobacterium rhizogenes A4 by a conventional method. Antisense-CS ginseng hairy roots were obtained by infecting the root discs of 3-year old P. ginseng with A. rhizogenes containing antisense-CS vector. The positive hairy root lines were screened on MS medium with kanamycin. Northern blot analyses were performed with 3 randomly-selected control hairy root lines (hairy root lines with empty pBI121 vector: C-2, C-4, C-7) and 5 antisense-CS hairy root lines (A-16, A-25, A-28, A-33, A-38). The results indicated that the antisense-CS hairy root lines can transcribe abundant antisense CS-RNA, while there were almost no antisense CS-RNA in control lines. Furthermore the sense CS-RNA levels of antisense lines were remarkably lower than those of control lines.
According to our observations, all antisense-CS lines and control lines presented the typical traits of hairy roots and had no morphological difference. This suggested that antisense suppression of CS had no effect on morphous of ginseng hairy roots. However, in liquid MS medium, the antisense lines exhibited a slower growth than control lines at early stage; but there was no obvious difference in the final biomass. This implied that antisense-CS manipulation had deleterious effects on hairy root growth during early days of the culture cycle; therefore it is persuasive that phytosterols play an important role in ginseng hairy roots growth during early stage of the culture cycle.
Moreover, total phytosterol, 2,3-oxidosqualene and total ginsenoside levels of antisense-CS hairy root lines and control lines cultured in MS liquid medium, were analyzed and compared at appropriate time points of the culture cycle. Total ginsenoside levels in all lines increased rapidly after day 15 and reached a plateau after day 25, suggesting that ginsenosides are primarily synthesized during the later stage of the culture cycle. Before day 15, total ginsenoside levels were comparable in antisense-CS lines and control lines. However, after day 20, except A-33 line, all antisense-CS lines exhibited higher total ginsenoside levels than control lines. It is noteworthy that the total ginsenoside levels were different among antisense-CS lines, of which the A-16 line had the highest total ginsenoside level (about 200% of that of control lines); however there was no obvious difference in total ginsenoside levels of control lines. The total phytosterol levels of antisense-CS lines were lower than those of control lines, implying that phytosterol biosynthesis was suppressed by antisense suppression of CS. In addition, the total phytosterol levels of all lines were relatively stable all through the culture cycle. The 2,3-oxidosqualene levels of control lines were stable during the culture cycle, while the 2,3-oxidosqualene levels of antisense lines were obviously higher at early stage than at the later stage of the culture cycle. The 2,3-oxidosqualene levels of antisense lines were higher than those of control lines at early stage of the culture cycle, but comparable at the later stage.
To further dissect the mechanism of the enhanced ginsenoside levels in antisense-CS lines, DS and CS enzyme activities in antisense and control lines were determined at day 20 of the culture cycle. The results indicated that all antisense lines, except A-33, exhibited higher DS enzyme activities than control lines, while the CS enzyme activities of antisense lines were lower than control lines. As DS and CS are the key enzymes for phytosterol and ginsenoside biosynthesis, respectively; the decrease of CS enzyme activity and increase of DS enzyme activity in antisense lines could primarily account for the decreased phytosterol levels and enhanced ginsenoside levels in antisense lines. The decreased CS enzyme activities in antisense lines should be caused by antisense suppression of CS; while the increased DS activities in antisense lines were possibly induced by the higher 2,3-oxidosqualene level at early stage during the culture cycle.
We can conclude from above analysis that antisense suppression of CS in P. ginseng hairy roots can lead to decreased CS enzyme activity, thus inhibite the biosynthesis of cycloartenol, from which phytosterols derive, and further suppress phytosterol biosynthesis from 2,3-oxidosqualene. As phytosterols and ginsenosides share the same precursor, 2,3-oxidosqualene, suppression of phytosterol synthesis led to more precursor available for ginsenoside biosynthesis, therefore resulted in enhanced ginsenoside levels in antisense-CS P. ginseng hairy roots. Additionally, antisense suppression of CS also led to higher enzyme activity of DS, the key enzyme for ginsenoside biosynthesis. This might be caused by the higher levels of 2,3-oxidosqualene, which is the substrate of DS, during the early stage of the culture cycle of ginseng hairy roots. Antisense suppression of CS had no effects on the morphous of P. ginseng hairy roots, while led to a slow growth rate of P. ginseng hairy roots during early stage of the culture cycle. This study is significant for further investigation of ginsenoside biosynthesis pathway and ginsenoside biosynthesis regulation.
引文
[1]唐斌,程绪菊,刘江,等.人参皂苷药理作用研究进展[J].西南军医, 2005, 7(03): 45-47.
[2]任跃英,丛林,官秀芝,等.人参资源现状及可持续发展战略[J].人参研究, 2003, 15(04): 9-10.
[3]金慧,周经纬,娄子恒.人参产业与区域经济发展[J].人参研究, 2004, 12(01): 2-6.
[4] Furuya T, Yoshikawa T, Orihara Y, et al. Saponin production in cell suspension cultures of Panax ginseng[J]. Planta Med, 1983, 48(6): 83-87.
[5] Liu C J and Hou S S. Regulation of fungal elicitors on the cell growth and biosynthesis of saponin of Panax ginseng cell suspension culture[J]. Shi Yan Sheng Wu Xue Bao, 1999, 32(2): 168-174.
[6] Thanh N T, Murthy H N, Yu K W, et al. Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors[J]. Appl Microbiol Biotechnol, 2005, 67(2): 197-201.
[7] Furuya T, Kojima H, Syono K, et al. Isolation of saponins and sapogenins from callus tissue of Panax ginseng[J]. Chem Pharm Bull (Tokyo), 1973, 21(1): 98-101.
[8] Furuya T, Yoshikawa T, Ishii T, et al. Regulation of saponin production in callus cultures of Panax ginseng[J]. Planta Med, 1983, 47(4): 200-204.
[9]蒋晶,王敬文.人参组织和细胞培养的研究Ⅱ.影响愈伤组织中人参皂甙生物合成的因素[J].林业科学研究, 1989, 2(02): 198-201.
[10]蒋晶,王敬文.人参组织和细胞培养的研究Ⅰ.环境因子对人参愈伤组织生长的影响[J].林业科学研究, 1988, 1989(06): 23-35.
[11] Kim Y S, Hahn E J, Murthy H N, et al. Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate[J]. Biotechnol Lett, 2004, 26(21): 1619-1622.
[12] Kim Y S, Yeung E C, Hahn E J, et al. Combined effects of phytohormone, indole-3-butyric acid, and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A. Meyer[J]. Biotechnol Lett, 2007, 29(11): 1789-1792.
[13] Yu K-W, Gaob W, Hahn E-J, et al. Jasmonic acid improves ginsenoside accumulation in adventitious root culture of Panax ginseng C.A. Meyer[J]. Biochemical Engineering Journal, 2002, 11: 211-215.
[14] Mallol A, Cusido R M, Palazon J, et al. Ginsenoside production in different phenotypes of Panax ginseng transformed roots[J]. Phytochemistry, 2001, 57(3): 365-371.
[15]赵寿经,李昌禹,钱延春,等.人参发根的诱导及其适宜培养条件的研究[J].生物工程学报, 2004, 20(02): 215-220.
[16]赵寿经,杨振堂,李昌禹,等.发根农杆菌A4菌株诱导人参根外植体产生人参发根及人参皂甙的生产方法:中国, 99111664[P]. 2001-02-28.
[17]赵寿经,杨振堂,李昌禹,等.发根农杆菌介导的人参遗传转化及人参皂甙工厂化生产[J].云南大学学报(自然科学版), 1999(S3): 34-37.
[18]孙彬贤,杨光孝,汪沁琳,等.人参毛状根合成人参皂苷培养条件的优化[J].中成药, 2003, 25(09): 746-748.
[19] Palazóna J, Cusidóa R, Bonfilla M, et al. Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production[J]. Plant Physiology and Biochemistry, 2003, 41(11-12): 1019-1025.
[20] Woo S S, Song J S, Lee J Y, et al. Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis[J]. Phytochemistry, 2004, 65(20): 2751-2761.
[21]周倩耘,丁家宜,刘峻,等.植物激素对人参毛状根生长和皂甙含量的影响[J].植物资源与环境学报, 2003, 12(01): 26-28.
[22]周倩耘,周建民,刘峻,等.诱导子对人参毛状根中皂苷含量的影响[J].南京军医学院学报, 2003, 25(02): 76-78.
[23] Haralampidis K, Trojanowska M, and Osbourn A E. Biosynthesis of triterpenoid saponins in plants[J]. Adv Biochem Eng Biotechnol, 2002, 75: 31-49.
[24] Lee M H, Jeong J H, Seo J W, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene[J]. Plant & Cell Physiology, 2004, 45(8): 976-984.
[25] Tansakul P, Shibuya M, Kushiro T, et al. Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng[J]. FEBS Letters, 2006, 580(22): 5143-5149.
[26] Kushiro T, Shibuya M, and Ebizuka Y. Beta-amyrin synthase--cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants[J]. European Journal of Biochemistry, 1998, 256(1): 238-244.
[27]王波,陈梅红.反义技术研究进展[J].中国生物工程杂志, 2004, 24(12): 43-47.
[28] Lee L K and Roth C M. Antisense technology in molecular and cellularbioengineering[J]. Curr Opin Biotechnol, 2003, 14(5): 505-511.
[29]柴晓杰,王丕武,关淑艳,等.反义技术及其在植物基因工程中的应用[J].吉林农业大学学报, 2004, 26(05): 515-518.
[30] Wang T W, Zhang C G, Wu W, et al. Antisense suppression of deoxyhypusine synthase in tomato delays fruit softening and alters growth and development[J]. Plant Physiol, 2005, 138(3): 1372-1382.
[31] Wang T-W, Wu1 W, and Zhang C-G. Antisense suppression of deoxyhypusine synthase by vacuum-infiltration of Agrobacterium enhances growth and seed yield of canola[J]. Physiologia Plantarum, 2005, 124: 493-503.
[32] Park S-U, Yu M, and Facchini P J. Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of california poppy[J]. Plant Physiol, 2002, 128(2): 696-706.
[33] Kiefer D and Pantuso T. Panax ginseng[J]. American Family Physician, 2003, 68(8): 1539-1542.
[34]张春枝,安利佳,金凤燮.人参皂苷生理活性的研究进展[J].食品与发酵工业, 2002, 25(04): 70-74.
[35]闫兆光.人参生产技术现状与发展对策[J].中药研究与信息, 2005, 7(11): 31-32.
[36]张中朋.人参出口现状及分析——人参十年出口回顾[J].中国现代中药, 2006, 8(07): 43-44.
[37]李刚,蔡天智.浅谈人参出口问题[J].中药研究与信息, 2003, 5(08): 59-61.
[38]张伟伟,王康胜,项华美.浅谈人参的规格、保管及加工[J].黑龙江中医药, 2004(04): 53.
[39]陈燕.鲜人参、生晒参和红参的比较研究[J].海峡药学, 2006, 18(04): 137-139.
[40]孔宪来,邢作山,邢作民,等.人参栽培加工留种技术[J].陕西农业科学, 2004(05): 111-113.
[41]白莉华,厉曙光,王力强,等. 3种不同人参胶囊对果蝇寿命的影响[J].上海预防医学杂志, 2006, 18(04): 157-160.
[42]邓金萍,杨万英,邓金荣,等.五合一人参油系列化妆品及其制备方法:中国, 92103846 [P]. 1993-12-08.
[43] Chang Y S, Seo E K, Gyllenhaal C, et al. Panax ginseng: a role in cancer therapy?[J]. Integr Cancer Ther, 2003, 2(1): 13-33.
[44] Shibata S. Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds[J]. Journal of Korean Medical Science, 2001, 16(6): 28-37.
[45] Helms S. Cancer prevention and therapeutics: Panax ginseng[J]. Alternative medicine review : a journal of clinical therapeutic, 2004, 9(3): 259-274.
[46] Guan S and Ge M. Experimental study on anti-fatigue effect of shenfu injection on diaphragmatic muscle[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2000, 20(5): 359-361.
[47] Wang B X, Cui J C, Liu A J, et al. Studies on the anti-fatigue effect of the saponins of stems and leaves of Panax ginseng (SSLG)[J]. J Tradit Chin Med, 1983, 3(2): 89-94.
[48] Park M W, Ha J, and Chung S H. 20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK[J]. Biol Pharm Bull, 2008, 31(4): 748-751.
[49] Banz W J, Iqbal M J, Bollaert M, et al. Ginseng modifies the diabetic phenotype and genes associated with diabetes in the male ZDF rat[J]. Phytomedicine, 2007, 14(10): 681-689.
[50] Lee W K, Kao S T, Liu I M, et al. Ginsenoside Rh2 is one of the active principles of Panax ginseng root to improve insulin sensitivity in fructose-rich chow-fed rats[J]. Horm Metab Res, 2007, 39(5): 347-54.
[51]傅晓晴,蔡宗苇,杨显荣.人参延缓中枢神经系统衰老的生理调节治疗作用的研究[J].中医药学刊, 2003, 21(07): 1044-1047.
[52] Cheng Y, Shen L H, and Zhang J T. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action[J]. Acta Pharmacol Sin, 2005, 26(2): 143-149.
[53] Liu M. Studies on the anti-aging and nootropic effects of ginsenoside Rg1 and its mechanisms of actions[J]. Sheng Li Ke Xue Jin Zhan, 1996, 27(2): 139-142.
[54] Rausch W D, Liu S, Gille G, et al. Neuroprotective effects of ginsenosides[J]. Acta Neurobiol Exp (Wars), 2006, 66(4): 369-375.
[55] Cheng Y, Shen L H, and Zhang J T. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action[J]. Acta Pharmacol Sin, 2005, 26(2): 143-149.
[56] Liu M. Studies on the anti-aging and nootropic effects of ginsenoside Rg1 and its mechanisms of actions[J]. Sheng Li Ke Xue Jin Zhan, 1996, 27(2): 139-142.
[57] Shen L H and Zhang J T. Ginsenoside Rg1 promotes proliferation of hippocampal progenitor cells[J]. Neurol Res, 2004, 26(4): 422-428.
[58] Chang M S, Lee S G, and Rho H M. Transcriptional activation of Cu/Zn superoxide dismutase and catalase genes by panaxadiol ginsenosides extracted from Panax ginseng[J]. Phytother Res, 1999, 13(8): 641-644.
[59] Liu Z, Luo X, and Liu G. In vitro study of the relationship between the structure of ginsenoside and its antioxidative or prooxidative activity in free radical induced hemolysis of human erythrocytes[J]. J Agric Food Chem 2003, 51: 2555-2558.
[60] Tian J, Fu F, Geng M, et al. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebral ischemia in rats[J]. Neurosci Lett, 2005, 374(2): 92-97.
[61] Kim J H, Cho S Y, Lee J H, et al. Neuroprotective effects of ginsenoside Rg3 against homocysteine-induced excitotoxicity in rat hippocampus[J]. Brain Res, 2007, 1136(1): 190-199.
[62] Leung K W, Yung K K, Mak N K, et al. Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity[J]. Neuropharmacology, 2007, 52(3): 827-835.
[63] Yuan Q L, Yang C X, Xu P, et al. Neuroprotective effects of ginsenoside Rb1 on transient cerebral ischemia in rats[J]. Brain Res, 2007, 1167: 1-12.
[64]张艳,人参皂苷Rg3的抗疲劳作用研究[D].长春:吉林大学生命科学院, 2007.
[65]王本祥,崔景朝,刘爱晶,等.人参茎叶皂甙抗疲劳作用的研究[J].中医杂志, 1981, 22(9): 697-699.
[66] Masuno H, Kitao T, and Okuda H. Ginsenosides increase secretion of lipoprotein lipase by 3T3-L1 adipocytes[J]. Biosci Biotechnol Biochem, 1996, 60(12): 1962-1965.
[67] Oh G S, Pae H O, Choi B M, et al. 20(S)-Protopanaxatriol, one of ginsenoside metabolites, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expressions through inactivation of nuclear factor-kappaB in RAW 264.7 macrophages stimulated with lipopolysaccharide[J]. Cancer Lett, 2004, 205(1): 23-29.
[68] Keum Y S, Han S S, Chun K S, et al. Inhibitory effects of the ginsenoside Rg3 on phorbol ester-induced cyclooxygenase-2 expression, NF-kappaB activation and tumor promotion[J]. Mutat Res, 2003, 523-524: 75-85.
[69] Matsuda H, Samukawa K, and Kubo M. Anti-inflammatory activity of ginsenoside Ro[J]. Planta Med, 1990, 56(1): 19-23.
[70] Park E K, Choo M K, Han M J, et al. Ginsenoside Rh1 possesses antiallergic and anti-inflammatory activities[J]. Int Arch Allergy Immunol, 2004, 133(2): 113-120.
[71] Zhou W and Zhou P. Advances in the study of ginsenoside compound K[J]. Yao Xue Xue Bao, 2007, 42(9): 917-923.
[72] Hwang J T, Kim S H, Lee M S, et al. Anti-obesity effects of ginsenoside Rh2 are associated with the activation of AMPK signaling pathway in 3T3-L1adipocyte[J]. Biochem Biophys Res Commun, 2007, 364(4): 1002-1008.
[73] Hwang J T, Lee M S, Kim H J, et al. Antiobesity effect of ginsenoside Rg3 involves the AMPK and PPAR-gamma signal pathways[J]. Phytother Res, 2009, 23(2): 262-266.
[74] Park M W, Ha J, and Chung S H. 20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK[J]. Biol Pharm Bull, 2008, 31(4): 748-751.
[75] Lee W K, Kao S T, Liu I M, et al. Increase of insulin secretion by ginsenoside Rh2 to lower plasma glucose in Wistar rats[J]. Clin Exp Pharmacol Physiol, 2006, 33(1-2): 27-32.
[76] Xie J T, Mehendale S R, Li X, et al. Anti-diabetic effect of ginsenoside Re in ob/ob mice[J]. Biochim Biophys Acta, 2005, 1740(3): 319-325.
[77] Liang Y and Zhao S. Progress in understanding of ginsenoside biosynthesis[J]. Plant Biol (Stuttg), 2008, 10(4): 415-421.
[78] Kim M K, Lee B S, In J G, et al. Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf[J]. Plant Cell Rep, 2006, 25(6): 599-606.
[79] Shin H R, Kim J Y, Yun T K, et al. The cancer-preventive potential of Panax ginseng: a review of human and experimental evidence[J]. Cancer Causes Control, 2000, 11(6): 565-576.
[80] Dou D, Li W, Guo N, et al. Ginsenoside Rg8, a new dammarane-type triterpenoid saponin from roots of Panax quinquefolium[J]. Chemical & Pharmaceutical Bulletin, 2006, 54(5): 751-753.
[81] Dou D Q, Chen Y J, Liang L H, et al. Six new dammarane-type triterpene saponins from the leaves of Panax ginseng[J]. Chemical & Pharmaceutical Bulletin, 2001, 49(4): 442-446.
[82] Park I H, Han S B, Kim J M, et al. Four new acetylated ginsenosides from processed ginseng (sun ginseng)[J]. Archives of Pharmacal Research, 2002a, 25(6): 837-841.
[83] Park I H, Kim N Y, Han S B, et al. Three new dammarane glycosides from heat processed ginseng[J]. Archives of Pharmacal Research, 2002b, 25(4): 428-432.
[84] Wang J Y, Li X G, Zheng Y N, et al. Isoginsenoside-Rh3, a new triterpenoid saponin from the fruits of Panax ginseng C.A. Mey[J]. Journal of Asian Natural Products Research, 2004, 6(4): 289-293.
[85] Yoshikawa M, Sugimoto S, Nakamura S, et al. Medicinal flowers. XI. Structures of new dammarane-type triterpene diglycosides with hydroperoxide group from flower buds of Panax ginseng[J]. Chemical & Pharmaceutical Bulletin, 2007, 55(4): 571-576.
[86] Abe I, Rohmer M, and Prestwich G D. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes[J]. Chemical Reviews, 1993, 93(6): 2189-2206.
[87] Szkopinska A, Swiezewska E, and Karst F. The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae[J]. Biochem Biophys Res Commun, 2000, 267(1): 473-477.
[88] Belles X, Martin D, and Piulachs M D. The mevalonate pathway and the synthesis of juvenile hormone in insects[J]. Annu Rev Entomol, 2005, 50: 181-199.
[89] Hoeffler J F, Hemmerlin A, Grosdemange-Billiard C, et al. Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate[J]. The Biochemical Journal, 2002, 366(Pt 2): 573-583.
[90] Lange B M and Croteau R. Isoprenoid biosynthesis via a mevalonate-independent pathway in plants: cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint[J]. Arch Biochem Biophys, 1999, 365(1): 170-174.
[91] Seemann M, Tse Sum Bui B, Wolff M, et al. Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG[J]. FEBS Letters, 2006, 580(6): 1547-1552.
[92] Rodriguez-Concepcion M and Boronat A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics[J]. Plant Physiology, 2002, 130(3): 1079-1089.
[93] Rohdich F, Zepeck F, Adam P, et al. The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(4): 1586-1591.
[94] Kushiro T, Ohno Y, Shibuya M, et al. In vitro conversion of 2,3-oxidosqualene into dammarenediol by Panax ginseng microsomes[J]. Biological & Pharmaceutical Bulletin, 1997, 20(3): 292-294.
[95] Jung J D, Park H W, Hahn Y, et al. Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags[J]. Plant Cell Reports, 2003, 22(3): 224-230.
[96] Seo J W, Jeong J H, Shin C G, et al. Overexpression of squalene synthase inEleutherococcus senticosus increases phytosterol and triterpene accumulation[J]. Phytochemistry, 2005, 66(8): 869-877.
[97] Choi D W, Jung J, Ha Y I, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites[J]. Plant Cell Reports, 2005, 23(8): 557-566.
[98] Kim M K, Lee B S, In J G, et al. Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf[J]. Plant Cell Reports, 2006, 25(6): 599-606.
[99] Phillips D R, Rasbery J M, Bartel B, et al. Biosynthetic diversity in plant triterpene cyclization[J]. Current Opinion in Plant Biology, 2006, 9(3): 305-314.
[100] Han J Y, Kwon Y S, Yang D C, et al. Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng[J]. Plant & Cell Physiology, 2006, 47(12): 1653-1662.
[101]罗志勇,刘水平,陆秋恒,等.人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析[J].生命科学研究, 2003, 7(04): 324-328.
[102]张崇禧,郑友兰,张春红,等.不同方法提取人参总皂苷工艺的优化研究[J].人参研究, 2003, 15(04): 5-8.
[103]张春红,张崇禧,郑友兰,等.浸渍法提取人参皂苷最佳工艺的研究[J].吉林农业大学学报, 2003, 25(01): 73-74.
[104]刘永练,郑成,毛桃嫣,等.微波提取人参中活性成分人参皂苷的研究[J].广州大学学报(自然科学版), 2006, 5(6): 52-55.
[105] Zhang S, Chen R, Wu H, et al. Ginsenoside extraction from Panax quinquefolium L. (American ginseng) root by using ultrahigh pressure[J]. J Pharm Biomed Anal, 2006, 41(1): 57-63.
[106]张晶,陈全成,弓晓杰,等.不同提取方法对人参皂苷提取率的影响[J].吉林农业大学学报, 2003, 25(01): 71-72.
[107]张宪臣,王淑敏,陈光,等.人参总皂苷超声提取工艺的研究[J].现代中药研究与实践, 2005, 19(06): 55-57.
[108]彭拓华,陈洁炜,黄湘兰,等.大孔树脂富集纯化人参总皂苷工艺条件优选[J].中药材, 2006, 29(04): 392-394.
[109]李洪刚,何克江,韩蔚,等.树脂联用纯化人参总皂苷[J].中医药学刊, 2005, 23(04): 708-711.
[110]桂双英,周亚球.比色法测定人参中人参总皂苷的含量[J].安徽中医学院学报, 2003, 22(04): 51-52.
[111]孔燕君,洪美凤.紫外分光光度法测定人参及三七中总皂苷含量[J].中国现代应用药学, 2000, 17(01): 51-52.
[112] Corthout J, Naessens T, Apers S, et al. Quantitative determination of ginsenosides from Panax ginseng roots and ginseng preparations by thin layer chromatography--densitometry[J]. J Pharm Biomed Anal, 1999, 21(1): 187-192.
[113]潘坚扬,程翼宇,王毅,等. 9种人参皂苷同时测定方法及在人参质量鉴别中的应用[J].分析化学, 2005, 33(11): 1565-1568.
[114] Chan T W, But P P, Cheng S W, et al. Differentiation and authentication of Panax ginseng, Panax quinquefolius, and ginseng products by using HPLC/MS[J]. Anal Chem, 2000, 72(6): 1281-1287.
[115] Fukuda N, Tanaka H, and Shoyama Y. Applications of ELISA, western blotting and immunoaffinity concentration for survey of ginsenosides in crude drugs of Panax species and traditional Chinese herbal medicines[J]. Analyst, 2000, 125(8): 1425-1429.
[116] Yoon S R, Nah J J, Kim S K, et al. Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay[J]. Chem Pharm Bull (Tokyo), 1998, 46(7): 1144-1147.
[117] Choi K, Kim M, Ryu J, et al. Ginsenosides compound K and Rh(2) inhibit tumor necrosis factor-alpha-induced activation of the NF-kappaB and JNK pathways in human astroglial cells[J]. Neurosci Lett, 2007, 421(1): 37-41.
[118] Bae E A, Han M J, Kim E J, et al. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants[J]. Arch Pharm Res, 2004, 27(1): 61-67.
[119] Cheng L Q, Kim M K, Lee J W, et al. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia[J]. Biotechnol Lett, 2006, 28(14): 1121-1127.
[120] Ko S R, Suzuki Y, Kim Y H, et al. Enzymatic synthesis of two ginsenoside Re-beta-xylosides[J]. Biosci Biotechnol Biochem, 2001, 65(5): 1223-1226.
[121] Ko S R, Suzuki Y, Choi K J, et al. Enzymatic preparation of genuine prosapogenin, 20(S)-ginsenoside Rh1, from ginsenosides Re and Rg1[J]. Biosci Biotechnol Biochem, 2000, 64(12): 2739-2743.
[122] Danieli B, Falcone L, Monti D, et al. Regioselective enzymatic glycosylation of natural polyhydroxylated compounds: galactosylation and glucosylation of protopanaxatriol ginsenosides[J]. J Org Chem, 2001, 66(1): 262-269.
[123] Wu J and Zhong J-J. Production of ginseng and its bioactive components in plant cell culture: Current technological and applied aspects[J]. Journal of Biotechnology, 1999, 68: 89-99.
[124] Lin L, Wu J, Ho K P, et al. Ultrasound-induced physiological effects and secondary metabolite (saponin) production in Panax ginseng cell cultures[J].Ultrasound Med Biol, 2001, 27(8): 1147-1152.
[125] Liu C J and Hou S S. Regulation of fungal elicitors on the cell growth and biosynthesis of saponin of Panax ginseng cell suspension culture[J]. Shi Yan Sheng Wu Xue Bao, 1999, 32(2): 168-174.
[126] Wu J and Ho K P. Assessment of various carbon sources and nutrient feeding strategies for Panax ginseng cell culture[J]. Appl Biochem Biotechnol, 1999, 82(1): 17-26.
[127] Thanh N T, Murthy H N, Yu K W, et al. Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng[J]. J Plant Physiol, 2006, 163(12): 1337-1341.
[128] Wu J Y, Wong K, Hoa K P, et al. Enhancement of saponin production in Panax ginseng cell culture by osmotic stress and nutrient feeding[J]. Enzyme and Microbial Technology, 2005, 36: 133-138.
[129] Yu K W, Murthy H N, Jeong C S, et al. Organic germanium stimulates the growth of ginseng adventitious roots and ginsenoside production [J]. Process Biochemistry 2005, 40: 2959–2961.
[130] Jeong C-S, Chakrabarty D, Hahna E-J, et al. Effects of oxygen, carbon dioxide and ethylene on growth and bioactive compound production in bioreactor culture of ginseng adventitious roots[J]. Biochemical Engineering Journal, 2006, 27: 252-263.
[131] Furuya T, Yoshikawa T, Ishii T, et al. Effects of auxins on growth and saponin production in callus cultures of Panax ginseng[J]. Planta Med, 1983, 47(3): 183-187.
[132] Furuya T, Yoshikawa T, Ishii T, et al. Regulation of saponin production in callus cultures of Panax ginseng[J]. Planta Med, 1983, 47(4): 200-204.
[133] Giri A and Narasu M L. Transgenic hairy roots: recent trends and applications[J]. Biotechnol Adv, 2000, 18(1): 1-22.
[134] Usami S, Morikawa S, Takebe I, et al. Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium[J]. Mol Gen Genet, 1987, 209(2): 221-226.
[135]张辉,于荣敏.发根农杆菌Ri质粒及其在植物次生代谢产物研究中的应用[J].沈阳药科大学学报, 2000, 17(04): 306-308.
[136] Hu Z-B and Du M. Hairy root and its application in plant genetic engineering[J]. Journal of Integrative Plant Biology 2006, 48(2): 121-127.
[137] Chilton M, Tepfer D, Petit A, et al. Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells.[J]. Nature, 1982, 295: 432-434.
[138] Ooms G, Twell D, Bossen M, et al. Development regulation of Ri T DNAgene expression in roots, shoots and tubers of transformed potato (Solanum tuberosum cv. Desiree)[J]. Plant Mol Biol, 1986, 6: 321-330.
[139] Stachel SE M E, Van Montagu M, Zambryski P. Identification of signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J]. Nature, 1985, 318: 624-629.
[140] Kumar V, Jones B, and Davey M. Transformation by Agrobacterium rhizogenes and regeneration of transgenic shoots of the wild soybean Glycine argyrea.[J]. Plant Cell Reports, 1991, 10: 135-138.
[141] Giri A, Banerjee S, Ahuja P, et al. Production of hairy roots in Aconitum heterophyllum wall using Agrobacterium rhizogenes[J]. In Vitro Cell Dev Biol-Plant, 1997, 33: 280-284.
[142] Nguyen C, Bourgaud F, Forlot P, et al. Establishment of hairy root cultures of Psoralea species[J]. Plant Cell Reports, 1992, 11: 424-427.
[143]张兴,刘晓娟,吕巧玲,等.毛状根生产次生代谢产物的研究进展[J].化工进展, 2007, 26(09): 1228-1232.
[144] Guillon S, Tremouillaux-Guiller J, Pati P K, et al. Harnessing the potential of hairy roots: dawn of a new era[J]. Trends Biotechnol, 2006, 24(9): 403-409.
[145] Sevon N, Drager B, Hiltunen R, et al. Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus[J]. Plant Cell Reports, 1997, 16: 605-611.
[146] Hoshino Y and Mii M. Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes[J]. Plant Cell Reports, 1998, 17: 256-261.
[147] Perez-Molphe E and Ochoa-Alejo N. Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes transformed tissues[J]. Plant Cell Reports, 1998, 17: 591-596.
[148] Handa T, Sujimura T, Kato E, et al. Genetic transformation of Eustoma grandiflorum with rol genes[J]. Acta Hort, 1995, 392: 209-218.
[149] Damiani F and Aricioni S. Transformation of Medicago arborea L. with Agrobacterium rhizogenes binary vector carrying the hygromycin resistance genes[J]. Plant Cell Reports, 1991(10): 300-303.
[150] Bourgaud F, Cravot A, Milesi S, et al. Production of plant secondary metabolites: a historical perspective[J]. Plant Science, 2001, 161: 839-851.
[151]张广求,王伯初,段传人,等.提高毛状根中次生代谢产物含量的方法与技术[J].重庆大学学报(自然科学版), 2005, 28(06): 121-124.
[152] Mateus L, Cherkaoui S, Christen P, et al. Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography[J].Phytochemistry, 2000, 54(5): 517-523.
[153] Pavlov A, Berkov S, Weber J, et al. Hyoscyamine biosynthesis in Datura stramonium hairy root in vitro systems with different ploidy levels[J]. Appl Biochem Biotechnol, 2008.
[154] Batra J, Dutta A, Singh D, et al. Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration[J]. Plant Cell Rep, 2004, 23(3): 148-154.
[155] Wu J Y, Ng J, Shi M, et al. Enhanced secondary metabolite (tanshinone) production of Salvia miltiorrhiza hairy roots in a novel root-bacteria coculture process[J]. Appl Microbiol Biotechnol, 2007, 77(3): 543-550.
[156] Morgan J A and Shanks J V. Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures[J]. J Biotechnol, 2000, 79(2): 137-145.
[157] Boitel-Conti M, Gontier E, Laberche J C, et al. Permeabilization of Datura innoxia hairy roots for release of stored tropane alkaloids[J]. Planta Med, 1995, 61(3): 287-290.
[158] Lee K, Yamakawa T, Kodama T, et al. Effects of chemicals on alkaloid production by transformed roots of Belladonna[J]. Phytochemistry, 1998, 49: 2343-2347.
[159] Yan Q, Hu Z, Tan R X, et al. Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation[J]. J Biotechnol, 2005, 119(4): 416-424.
[160] Rudrappa T, Neelwarne B, and Aswathanarayana R G.. In situ and Ex situ adsorption and recovery of betalains from hairy root cultures of Beta vulgaris[J]. Biotechnol Prog, 2004, 20(3): 777-785.
[161] Bais H P, Govindaswamy S, and Ravishankar G A. Enhancement of growth and coumarin production in hairy root cultures of witloof chicory (Cichorium intybus L.cv. Lucknow local) under the influence of fungal elicitors[J]. J Biosci Bioeng, 2000, 90(6): 648-653.
[162] Bhagwath S G and Hjortso M A. Statistical analysis of elicitation strategies for thiarubrine A production in hairy root cultures of Ambrosia artemisiifolia[J]. J Biotechnol, 2000, 80(2): 159-167.
[163] Chen H, Chena F, Chiu F C, et al. The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza[J]. Enzyme Microb Technol, 2001, 28(1): 100-105.
[164] Jeong G T, Park D H, Ryu H W, et al. Production of antioxidant compoundsby culture of Panax ginseng C.A. Meyer hairy roots: I. Enhanced production of secondary metabolite in hairy root cultures by elicitation[J]. Appl Biochem Biotechnol, 2005, 121-124: 1147-1157.
[165] Kim O T, Bang K H, Shin Y S, et al. Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate[J]. Plant Cell Rep, 2007, 26(11): 1941-1949.
[166] Komaraiah P, Reddy G V, Reddy P S, et al. Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum[J]. Biotechnol Lett, 2003, 25(8): 593-597.
[167] Pitta-Alvarez S I, Spollansky T C, and Giulietti A M. The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida[J]. Enzyme Microb Technol, 2000, 26(2-4): 252-258.
[168] Rijhwani S K and Shanks J V. Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures[J]. Biotechnol Prog, 1998, 14(3): 442-449.
[169] Magnotta M, Murata J, Chen J, et al. Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots[J]. Phytochemistry, 2007, 68(14): 1922-1931.
[170] Hughes E H, Hong S B, Gibson S I, et al. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine[J]. Metab Eng, 2004, 6(4): 268-276.
[171] Hong S B, Peebles C A, Shanks J V, et al. Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots[J]. J Biotechnol, 2006, 122(1): 28-38.
[172] Menzel G, Harloff H J, and Jung C. Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.)[J]. Appl Microbiol Biotechnol, 2003, 60(5): 571-576.
[173] Franks T K, Powell K S, Choimes S, et al. Consequences of transferring three sorghum genes for secondary metabolite (cyanogenic glucoside) biosynthesis to grapevine hairy roots[J]. Transgenic Res, 2006, 15(2): 181-195.
[174] Li X-Q, Tan A, Voegtline M, et al. Expression of Cry5B protein from Bacillus thuringiensis in plant roots confers resistance to root-knot nematode[J]. Biological Control, 2008, 47: 97–102.
[175] Gaume A, Komarnytsky S, Borisjuk N, et al. Rhizosecretion of recombinant proteins from plant hairy roots[J]. Plant Cell Rep, 2003, 21(12): 1188-1193.
[176] Shi H P and Lindemann P. Expression of recombinant Digitalis lanata EHRH. cardenolide 16'-O-glucohydrolase in Cucumis sativus L. hairy roots[J]. Plant Cell Rep, 2006, 25(11): 1193-1198.
[177] Kumar G B S, Ganapathi T R, Srinivas L, et al. Expression of hepatitis B surface antigen in potato hairy roots[J]. Plant Science, 2006, 170: 918–925.
[178] Jin U H, Chun J A, Lee J W, et al. Expression and characterization of extracellular fungal phytase in transformed sesame hairy root cultures[J]. Protein Expr Purif, 2004, 37(2): 486-492.
[179] Pletsch M, de Araujo B S, and Charlwood B V. Novel biotechnological approaches in environmental remediation research[J]. Biotechnol Adv, 1999, 17(8): 679-687.
[180] Gonz′alez P S, Capozucca C E, Tigierm H A, et al. Phytoremediation of phenol from wastewater, by peroxidases of tomato hairy root cultures[J]. Enzyme and Microbial Technology, 2006, 39: 647-653.
[181] Coniglio M S, Busto V D, Gonzalez P S, et al. Application of Brassica napus hairy root cultures for phenol removal from aqueous solutions[J]. Chemosphere, 2008, 72(7): 1035-1042.
[182] de Araujo B S, Dec J, Bollag J M, et al. Uptake and transformation of phenol and chlorophenols by hairy root cultures of Daucus carota, Ipomoea batatas and Solanum aviculare[J]. Chemosphere, 2006, 63(4): 642-651.
[183] Gonzalez P S, Agostini E, and Milrad S R. Comparison of the removal of 2,4-dichlorophenol and phenol from polluted water, by peroxidases from tomato hairy roots, and protective effect of polyethylene glycol[J]. Chemosphere, 2008, 70(6): 982-989.
[184] Rezek J, Macek T, Mackova M, et al. Plant metabolites of polychlorinated biphenyls in hairy root culture of black nightshade Solanum nigrum SNC-9O[J]. Chemosphere, 2007, 69(8): 1221-1227.
[185] Suresh B, Sherkhane P D, Kale S, et al. Uptake and degradation of DDT by hairy root cultures of Cichorium intybus and Brassica juncea[J]. Chemosphere, 2005, 61(9): 1288-1292.
[186] Nedelkoska T V and Doran P M. Hyperaccumulation of nickel by hairy roots of alyssum species: comparison with whole regenerated plants[J]. Biotechnol Prog, 2001, 17(4): 752-759.
[187] Nedelkoska T V and Doran P M. Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens[J]. Biotechnol Bioeng, 2000, 67(5): 607-615.
[188] Casas D A, Pitta-Alvarez S I, and Giulietti A M. Biotransformation ofhydroquinone by hairy roots of Brugmansia candida and effect of sugars and free-radical scavengers[J]. Appl Biochem Biotechnol, 1998, 69(2): 127-136.
[189] Yan C Y, Zhang Z, Yu R M, et al. Studies on biotransformation of arbutin by 4-hydroxy phenol in hairy root of Polygonum multiflorum[J]. Zhongguo Zhong Yao Za Zhi, 2007, 32(3): 192-195.
[190] Asada Y, Saito H, Yoshikawa T, et al. Biotransformation of 18 beta-glycyrrhetinic acid by ginseng hairy root culture[J]. Phytochemistry, 1993, 34(4): 1049-1052.
[191] Kanho H, Yaoya S, Kawahara N, et al. Biotransformation of benzaldehyde-type and acetophenone-type derivatives by Pharbitis nil hairy roots[J]. Chem Pharm Bull (Tokyo), 2005, 53(4): 361-365.
[192] Kwok K and Doran P. Kinetic and stoichiometric analysis of hairy roots in a segmented bubble column reactor[J]. Biotechnol Prog 1995, 11: 429-435.
[193]高文远,贾伟,段宏泉,等.药用植物发酵培养的工业化探讨[J].中国中药杂志, 2003, 28(05): 385-390.
[194] Kim Y, Wyslouzil B E, and Weathers P J. Secondary metabolism of hairy root cultures in bioreactors[J]. In Vitro Cell. Dev. Biol.-Plant, 2002, 38: 1-10.
[195] Hilton M G and Rhodes M J. Growth and hyoscyamine production of 'hairy root' cultures of Datura stramonium in a modified stirred tank reactor[J]. Appl Microbiol Biotechnol, 1990, 33(2): 132-138.
[196] Martin Y and Vermette P. Bioreactors for tissue mass culture: design, characterization, and recent advances[J]. Biomaterials, 2005, 26(35): 7481-7503.
[197]陈岺曦,王伯初.生物反应器与药用植物毛状根的大规模培养[J].生物技术通报, 2007(04): 38-41.
[198] Jeong G T, Park D H, Hwang B, et al. Studies on mass production of transformed Panax ginseng hairy roots in bioreactor[J]. Appl Biochem Biotechnol, 2002, 98-100: 1115-1127.
[199] Taya M, Yoyama A, Nomura R, et al. Production of peroxidase with horseradish hairy root cells in a two step culture system[J]. J Ferment Bioeng, 1989, 67: 31-34.
[200] Muranaka T, Ohakawa H, and Yamada Y. Scopolamine release into media by Duboisia leichhardtti hairy root clones[J]. Appl Microbiol Biotechnol, 1992, 37: 554-559.
[201] Kintzios S, Makri O, Pistola E, et al. Scale-up production of puerarin from hairy roots of Pueraria phaseoloides in an airlift bioreactor[J]. Biotechnol Lett, 2004, 26(13): 1057-1059.
[202] Peraza-Luna F, Rodriguez-Mendiola M, Arias-Castro C, et al. Sotoloneproduction by hairy root cultures of Trigonella foenum-graecum in airlift with mesh bioreactors[J]. J Agric Food Chem, 2001, 49(12): 6012-6019.
[203] Tescione L D, Ramakrishnan D, and Curtis W R. The role of liquid mixing and gas-phase dispersion in a submerged, sparged root reactor[J]. Enzyme Microb Technol, 1997, 20(3): 207-213.
[204] Pavlov A, Georgiev M, and Bley T. Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor[J]. Z Naturforsch [C], 2007, 62(5-6): 439-446.
[205] Souret F F, Kim Y, Wyslouzil B E, et al. Scale-up of Artemisia annua L. hairy root cultures produces complex patterns of terpenoid gene expression[J]. Biotechnol Bioeng, 2003, 83(6): 653-667.
[206] Woo S and Park J. Root culture using a mist culture system and estimation of scale-up feasibility[J]. J Chem Tech Biotechnol, 1996, 66: 355-362.
[207] Wyslouzil B E, Waterbury R G, and Weathers P J. The growth of single roots of Artemisia annua in nutrient mist reactors[J]. Biotechnol Bioeng, 2000, 70(2): 143-150.
[208] Kim, Kim Y, Wyslouzil, et al. A comparative study of mist and bubble column reactors in the in vitro production of artemisinin[J]. Plant Cell Reports, 2001, 20(5): 451-455.
[209] Uozumi N. Large-scale production of hairy root[J]. Adv Biochem Eng Biotechnol, 2004, 91: 75-103.
[210] Suresh B, Rajasekaran T, and Ramachandra S. Studies on osmolarity, conductivity and mass transfer for selection of a bioreactor for Tagetes patula L.hairy roots[J]. Process Biochemistry, 2001, 36(10): 987-993.
[211]唐正义,胡蓉.反义RNA研究进展[J].内江师范学院学报, 2004, 19(04): 30-35.
[212] Brantl S. Antisense-RNA regulation and RNA interference[J]. Biochim Biophys Acta, 2002, 1575(1-3): 15-25.
[213] Tomizawa J and Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA1 with primer transeript [J]. Proc Natl Acad Sci USA, 1981, 78: 1421-1425.
[214]娄虹,杨淑琴.反义RNA技术及其在农业领域的应用[J].辽宁大学学报(自然科学版), 2005, 32(04): 328-333.
[215]李伟明,张寒霜,赵俊丽,等.反义RNA技术及其在植物种质创新领域的应用.中国棉花学会2006年年会暨第七次代表大会论文汇编[C].保定: [出版者不祥],2006.
[216] Krol A and Mol J. Antisense genes in plants: an overview [J]. Gene, 1988, 72: 5-10.
[217] Oeller P, Iu M, and Tanlor P. Reversible inhibition of tomato fruit senescence by antisense RNA[J]. Science, 1991, 254: 437-439.
[218] Amemiya T, Kanayama Y, Yamaki S, et al. Fruit-specific V-ATPase suppression in antisense-transgenic tomato reduces fruit growth and seed formation[J]. Planta, 2006, 223(6): 1272-1280.
[219] Day A G, Bejarano E R, Buck K W, et al. Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus[J]. Proc Natl Acad Sci U S A, 1991, 88(15): 6721-6725.
[220] Bendahmane M and Gronenborn B. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA[J]. Plant Mol Biol, 1997, 33(2): 351-357.
[221] de Feyter R, Young M, Schroeder K, et al. A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco[J]. Mol Gen Genet, 1996, 250(3): 329-338.
[222] Levis C, Tronchet M, Meyer M, et al. Effects of antisense oligodeoxynucleotide hybridization on in vitro translation of potato virus Y RNA[J]. Virus Genes, 1992, 6(1): 33-46.
[223] Zhang P, Vanderschuren H, Futterer J, et al. Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes[J]. Plant Biotechnol J, 2005, 3(4): 385-397.
[224] Yui R, Iketani S, Mikami T, et al. Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility[J]. Plant J, 2003, 34(1): 57-66.
[225] Zhang Y, Shewry P R, Jones H, et al. Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley[J]. Plant J, 2001, 28(4): 431-441.
[226] Schmulling T, Rohrig H, Pilz S, et al. Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants[J]. Mol Gen Genet, 1993, 237(3): 385-394.
[227]张新宇,高燕宁. PCR引物设计及软件使用技巧[J].生物信息学, 2004(04): 1-8.
[228] Skrypina N A, Timofeeva A V, Khaspekov G L, et al. Total RNA suitable for molecular biology analysis[J]. J Biotechnol, 2003, 105(1-2): 1-9.
[229]王磊,范云六.植物微小RNA(microRNA)研究进展[J].中国农业科技导报, 2007, 9(3): 18-23.
[230]高文,贾洪涛.正义链还是反义链[J].临沂师范学院学报, 2003, 25(03): 62-63.
[231] Yoshikawa T and Furuya T. Saponin production by cultures of Panaxginseng transformed with Agrobacterium rhizogenes[J]. Plant Cell Reports, 1987, 6(6): 449-453.
[232]赵艳,于彦春.植物中转基因的整合机制[J].中国生物工程杂志, 2003, 23(2): 29-32.
[233] Shinozaki J, Shibuya M, Masuda K, et al. Squalene cyclase and oxidosqualene cyclase from a fern[J]. FEBS Lett, 2008, 582(2): 310-318.
[234]高瑜莹,裘爱泳,潘秋琴,等.植物甾醇的分析方法[J].中国油脂, 2001, 26(1): 25-28.
[235] Flores-Sanchez I, Ortega-Lopez J, Montes-Horcasitas M, et al. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.[J]. Plant Cell Physiol, 2002, 43: 1502-1509.
[236] Brian M, Carl A, Aideen H. Compositions with increased phytosterol levels obtained from plants with decreased triterpene saponin levels: US, 20070107081[P]. 2007-05-10.
[2]任跃英,丛林,官秀芝,等.人参资源现状及可持续发展战略[J].人参研究, 2003, 15(04): 9-10.
[3]金慧,周经纬,娄子恒.人参产业与区域经济发展[J].人参研究, 2004, 12(01): 2-6.
[4] Furuya T, Yoshikawa T, Orihara Y, et al. Saponin production in cell suspension cultures of Panax ginseng[J]. Planta Med, 1983, 48(6): 83-87.
[5] Liu C J and Hou S S. Regulation of fungal elicitors on the cell growth and biosynthesis of saponin of Panax ginseng cell suspension culture[J]. Shi Yan Sheng Wu Xue Bao, 1999, 32(2): 168-174.
[6] Thanh N T, Murthy H N, Yu K W, et al. Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors[J]. Appl Microbiol Biotechnol, 2005, 67(2): 197-201.
[7] Furuya T, Kojima H, Syono K, et al. Isolation of saponins and sapogenins from callus tissue of Panax ginseng[J]. Chem Pharm Bull (Tokyo), 1973, 21(1): 98-101.
[8] Furuya T, Yoshikawa T, Ishii T, et al. Regulation of saponin production in callus cultures of Panax ginseng[J]. Planta Med, 1983, 47(4): 200-204.
[9]蒋晶,王敬文.人参组织和细胞培养的研究Ⅱ.影响愈伤组织中人参皂甙生物合成的因素[J].林业科学研究, 1989, 2(02): 198-201.
[10]蒋晶,王敬文.人参组织和细胞培养的研究Ⅰ.环境因子对人参愈伤组织生长的影响[J].林业科学研究, 1988, 1989(06): 23-35.
[11] Kim Y S, Hahn E J, Murthy H N, et al. Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate[J]. Biotechnol Lett, 2004, 26(21): 1619-1622.
[12] Kim Y S, Yeung E C, Hahn E J, et al. Combined effects of phytohormone, indole-3-butyric acid, and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A. Meyer[J]. Biotechnol Lett, 2007, 29(11): 1789-1792.
[13] Yu K-W, Gaob W, Hahn E-J, et al. Jasmonic acid improves ginsenoside accumulation in adventitious root culture of Panax ginseng C.A. Meyer[J]. Biochemical Engineering Journal, 2002, 11: 211-215.
[14] Mallol A, Cusido R M, Palazon J, et al. Ginsenoside production in different phenotypes of Panax ginseng transformed roots[J]. Phytochemistry, 2001, 57(3): 365-371.
[15]赵寿经,李昌禹,钱延春,等.人参发根的诱导及其适宜培养条件的研究[J].生物工程学报, 2004, 20(02): 215-220.
[16]赵寿经,杨振堂,李昌禹,等.发根农杆菌A4菌株诱导人参根外植体产生人参发根及人参皂甙的生产方法:中国, 99111664[P]. 2001-02-28.
[17]赵寿经,杨振堂,李昌禹,等.发根农杆菌介导的人参遗传转化及人参皂甙工厂化生产[J].云南大学学报(自然科学版), 1999(S3): 34-37.
[18]孙彬贤,杨光孝,汪沁琳,等.人参毛状根合成人参皂苷培养条件的优化[J].中成药, 2003, 25(09): 746-748.
[19] Palazóna J, Cusidóa R, Bonfilla M, et al. Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production[J]. Plant Physiology and Biochemistry, 2003, 41(11-12): 1019-1025.
[20] Woo S S, Song J S, Lee J Y, et al. Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis[J]. Phytochemistry, 2004, 65(20): 2751-2761.
[21]周倩耘,丁家宜,刘峻,等.植物激素对人参毛状根生长和皂甙含量的影响[J].植物资源与环境学报, 2003, 12(01): 26-28.
[22]周倩耘,周建民,刘峻,等.诱导子对人参毛状根中皂苷含量的影响[J].南京军医学院学报, 2003, 25(02): 76-78.
[23] Haralampidis K, Trojanowska M, and Osbourn A E. Biosynthesis of triterpenoid saponins in plants[J]. Adv Biochem Eng Biotechnol, 2002, 75: 31-49.
[24] Lee M H, Jeong J H, Seo J W, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene[J]. Plant & Cell Physiology, 2004, 45(8): 976-984.
[25] Tansakul P, Shibuya M, Kushiro T, et al. Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng[J]. FEBS Letters, 2006, 580(22): 5143-5149.
[26] Kushiro T, Shibuya M, and Ebizuka Y. Beta-amyrin synthase--cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants[J]. European Journal of Biochemistry, 1998, 256(1): 238-244.
[27]王波,陈梅红.反义技术研究进展[J].中国生物工程杂志, 2004, 24(12): 43-47.
[28] Lee L K and Roth C M. Antisense technology in molecular and cellularbioengineering[J]. Curr Opin Biotechnol, 2003, 14(5): 505-511.
[29]柴晓杰,王丕武,关淑艳,等.反义技术及其在植物基因工程中的应用[J].吉林农业大学学报, 2004, 26(05): 515-518.
[30] Wang T W, Zhang C G, Wu W, et al. Antisense suppression of deoxyhypusine synthase in tomato delays fruit softening and alters growth and development[J]. Plant Physiol, 2005, 138(3): 1372-1382.
[31] Wang T-W, Wu1 W, and Zhang C-G. Antisense suppression of deoxyhypusine synthase by vacuum-infiltration of Agrobacterium enhances growth and seed yield of canola[J]. Physiologia Plantarum, 2005, 124: 493-503.
[32] Park S-U, Yu M, and Facchini P J. Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of california poppy[J]. Plant Physiol, 2002, 128(2): 696-706.
[33] Kiefer D and Pantuso T. Panax ginseng[J]. American Family Physician, 2003, 68(8): 1539-1542.
[34]张春枝,安利佳,金凤燮.人参皂苷生理活性的研究进展[J].食品与发酵工业, 2002, 25(04): 70-74.
[35]闫兆光.人参生产技术现状与发展对策[J].中药研究与信息, 2005, 7(11): 31-32.
[36]张中朋.人参出口现状及分析——人参十年出口回顾[J].中国现代中药, 2006, 8(07): 43-44.
[37]李刚,蔡天智.浅谈人参出口问题[J].中药研究与信息, 2003, 5(08): 59-61.
[38]张伟伟,王康胜,项华美.浅谈人参的规格、保管及加工[J].黑龙江中医药, 2004(04): 53.
[39]陈燕.鲜人参、生晒参和红参的比较研究[J].海峡药学, 2006, 18(04): 137-139.
[40]孔宪来,邢作山,邢作民,等.人参栽培加工留种技术[J].陕西农业科学, 2004(05): 111-113.
[41]白莉华,厉曙光,王力强,等. 3种不同人参胶囊对果蝇寿命的影响[J].上海预防医学杂志, 2006, 18(04): 157-160.
[42]邓金萍,杨万英,邓金荣,等.五合一人参油系列化妆品及其制备方法:中国, 92103846 [P]. 1993-12-08.
[43] Chang Y S, Seo E K, Gyllenhaal C, et al. Panax ginseng: a role in cancer therapy?[J]. Integr Cancer Ther, 2003, 2(1): 13-33.
[44] Shibata S. Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds[J]. Journal of Korean Medical Science, 2001, 16(6): 28-37.
[45] Helms S. Cancer prevention and therapeutics: Panax ginseng[J]. Alternative medicine review : a journal of clinical therapeutic, 2004, 9(3): 259-274.
[46] Guan S and Ge M. Experimental study on anti-fatigue effect of shenfu injection on diaphragmatic muscle[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2000, 20(5): 359-361.
[47] Wang B X, Cui J C, Liu A J, et al. Studies on the anti-fatigue effect of the saponins of stems and leaves of Panax ginseng (SSLG)[J]. J Tradit Chin Med, 1983, 3(2): 89-94.
[48] Park M W, Ha J, and Chung S H. 20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK[J]. Biol Pharm Bull, 2008, 31(4): 748-751.
[49] Banz W J, Iqbal M J, Bollaert M, et al. Ginseng modifies the diabetic phenotype and genes associated with diabetes in the male ZDF rat[J]. Phytomedicine, 2007, 14(10): 681-689.
[50] Lee W K, Kao S T, Liu I M, et al. Ginsenoside Rh2 is one of the active principles of Panax ginseng root to improve insulin sensitivity in fructose-rich chow-fed rats[J]. Horm Metab Res, 2007, 39(5): 347-54.
[51]傅晓晴,蔡宗苇,杨显荣.人参延缓中枢神经系统衰老的生理调节治疗作用的研究[J].中医药学刊, 2003, 21(07): 1044-1047.
[52] Cheng Y, Shen L H, and Zhang J T. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action[J]. Acta Pharmacol Sin, 2005, 26(2): 143-149.
[53] Liu M. Studies on the anti-aging and nootropic effects of ginsenoside Rg1 and its mechanisms of actions[J]. Sheng Li Ke Xue Jin Zhan, 1996, 27(2): 139-142.
[54] Rausch W D, Liu S, Gille G, et al. Neuroprotective effects of ginsenosides[J]. Acta Neurobiol Exp (Wars), 2006, 66(4): 369-375.
[55] Cheng Y, Shen L H, and Zhang J T. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action[J]. Acta Pharmacol Sin, 2005, 26(2): 143-149.
[56] Liu M. Studies on the anti-aging and nootropic effects of ginsenoside Rg1 and its mechanisms of actions[J]. Sheng Li Ke Xue Jin Zhan, 1996, 27(2): 139-142.
[57] Shen L H and Zhang J T. Ginsenoside Rg1 promotes proliferation of hippocampal progenitor cells[J]. Neurol Res, 2004, 26(4): 422-428.
[58] Chang M S, Lee S G, and Rho H M. Transcriptional activation of Cu/Zn superoxide dismutase and catalase genes by panaxadiol ginsenosides extracted from Panax ginseng[J]. Phytother Res, 1999, 13(8): 641-644.
[59] Liu Z, Luo X, and Liu G. In vitro study of the relationship between the structure of ginsenoside and its antioxidative or prooxidative activity in free radical induced hemolysis of human erythrocytes[J]. J Agric Food Chem 2003, 51: 2555-2558.
[60] Tian J, Fu F, Geng M, et al. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebral ischemia in rats[J]. Neurosci Lett, 2005, 374(2): 92-97.
[61] Kim J H, Cho S Y, Lee J H, et al. Neuroprotective effects of ginsenoside Rg3 against homocysteine-induced excitotoxicity in rat hippocampus[J]. Brain Res, 2007, 1136(1): 190-199.
[62] Leung K W, Yung K K, Mak N K, et al. Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity[J]. Neuropharmacology, 2007, 52(3): 827-835.
[63] Yuan Q L, Yang C X, Xu P, et al. Neuroprotective effects of ginsenoside Rb1 on transient cerebral ischemia in rats[J]. Brain Res, 2007, 1167: 1-12.
[64]张艳,人参皂苷Rg3的抗疲劳作用研究[D].长春:吉林大学生命科学院, 2007.
[65]王本祥,崔景朝,刘爱晶,等.人参茎叶皂甙抗疲劳作用的研究[J].中医杂志, 1981, 22(9): 697-699.
[66] Masuno H, Kitao T, and Okuda H. Ginsenosides increase secretion of lipoprotein lipase by 3T3-L1 adipocytes[J]. Biosci Biotechnol Biochem, 1996, 60(12): 1962-1965.
[67] Oh G S, Pae H O, Choi B M, et al. 20(S)-Protopanaxatriol, one of ginsenoside metabolites, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expressions through inactivation of nuclear factor-kappaB in RAW 264.7 macrophages stimulated with lipopolysaccharide[J]. Cancer Lett, 2004, 205(1): 23-29.
[68] Keum Y S, Han S S, Chun K S, et al. Inhibitory effects of the ginsenoside Rg3 on phorbol ester-induced cyclooxygenase-2 expression, NF-kappaB activation and tumor promotion[J]. Mutat Res, 2003, 523-524: 75-85.
[69] Matsuda H, Samukawa K, and Kubo M. Anti-inflammatory activity of ginsenoside Ro[J]. Planta Med, 1990, 56(1): 19-23.
[70] Park E K, Choo M K, Han M J, et al. Ginsenoside Rh1 possesses antiallergic and anti-inflammatory activities[J]. Int Arch Allergy Immunol, 2004, 133(2): 113-120.
[71] Zhou W and Zhou P. Advances in the study of ginsenoside compound K[J]. Yao Xue Xue Bao, 2007, 42(9): 917-923.
[72] Hwang J T, Kim S H, Lee M S, et al. Anti-obesity effects of ginsenoside Rh2 are associated with the activation of AMPK signaling pathway in 3T3-L1adipocyte[J]. Biochem Biophys Res Commun, 2007, 364(4): 1002-1008.
[73] Hwang J T, Lee M S, Kim H J, et al. Antiobesity effect of ginsenoside Rg3 involves the AMPK and PPAR-gamma signal pathways[J]. Phytother Res, 2009, 23(2): 262-266.
[74] Park M W, Ha J, and Chung S H. 20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK[J]. Biol Pharm Bull, 2008, 31(4): 748-751.
[75] Lee W K, Kao S T, Liu I M, et al. Increase of insulin secretion by ginsenoside Rh2 to lower plasma glucose in Wistar rats[J]. Clin Exp Pharmacol Physiol, 2006, 33(1-2): 27-32.
[76] Xie J T, Mehendale S R, Li X, et al. Anti-diabetic effect of ginsenoside Re in ob/ob mice[J]. Biochim Biophys Acta, 2005, 1740(3): 319-325.
[77] Liang Y and Zhao S. Progress in understanding of ginsenoside biosynthesis[J]. Plant Biol (Stuttg), 2008, 10(4): 415-421.
[78] Kim M K, Lee B S, In J G, et al. Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf[J]. Plant Cell Rep, 2006, 25(6): 599-606.
[79] Shin H R, Kim J Y, Yun T K, et al. The cancer-preventive potential of Panax ginseng: a review of human and experimental evidence[J]. Cancer Causes Control, 2000, 11(6): 565-576.
[80] Dou D, Li W, Guo N, et al. Ginsenoside Rg8, a new dammarane-type triterpenoid saponin from roots of Panax quinquefolium[J]. Chemical & Pharmaceutical Bulletin, 2006, 54(5): 751-753.
[81] Dou D Q, Chen Y J, Liang L H, et al. Six new dammarane-type triterpene saponins from the leaves of Panax ginseng[J]. Chemical & Pharmaceutical Bulletin, 2001, 49(4): 442-446.
[82] Park I H, Han S B, Kim J M, et al. Four new acetylated ginsenosides from processed ginseng (sun ginseng)[J]. Archives of Pharmacal Research, 2002a, 25(6): 837-841.
[83] Park I H, Kim N Y, Han S B, et al. Three new dammarane glycosides from heat processed ginseng[J]. Archives of Pharmacal Research, 2002b, 25(4): 428-432.
[84] Wang J Y, Li X G, Zheng Y N, et al. Isoginsenoside-Rh3, a new triterpenoid saponin from the fruits of Panax ginseng C.A. Mey[J]. Journal of Asian Natural Products Research, 2004, 6(4): 289-293.
[85] Yoshikawa M, Sugimoto S, Nakamura S, et al. Medicinal flowers. XI. Structures of new dammarane-type triterpene diglycosides with hydroperoxide group from flower buds of Panax ginseng[J]. Chemical & Pharmaceutical Bulletin, 2007, 55(4): 571-576.
[86] Abe I, Rohmer M, and Prestwich G D. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes[J]. Chemical Reviews, 1993, 93(6): 2189-2206.
[87] Szkopinska A, Swiezewska E, and Karst F. The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae[J]. Biochem Biophys Res Commun, 2000, 267(1): 473-477.
[88] Belles X, Martin D, and Piulachs M D. The mevalonate pathway and the synthesis of juvenile hormone in insects[J]. Annu Rev Entomol, 2005, 50: 181-199.
[89] Hoeffler J F, Hemmerlin A, Grosdemange-Billiard C, et al. Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate[J]. The Biochemical Journal, 2002, 366(Pt 2): 573-583.
[90] Lange B M and Croteau R. Isoprenoid biosynthesis via a mevalonate-independent pathway in plants: cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint[J]. Arch Biochem Biophys, 1999, 365(1): 170-174.
[91] Seemann M, Tse Sum Bui B, Wolff M, et al. Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG[J]. FEBS Letters, 2006, 580(6): 1547-1552.
[92] Rodriguez-Concepcion M and Boronat A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics[J]. Plant Physiology, 2002, 130(3): 1079-1089.
[93] Rohdich F, Zepeck F, Adam P, et al. The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(4): 1586-1591.
[94] Kushiro T, Ohno Y, Shibuya M, et al. In vitro conversion of 2,3-oxidosqualene into dammarenediol by Panax ginseng microsomes[J]. Biological & Pharmaceutical Bulletin, 1997, 20(3): 292-294.
[95] Jung J D, Park H W, Hahn Y, et al. Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags[J]. Plant Cell Reports, 2003, 22(3): 224-230.
[96] Seo J W, Jeong J H, Shin C G, et al. Overexpression of squalene synthase inEleutherococcus senticosus increases phytosterol and triterpene accumulation[J]. Phytochemistry, 2005, 66(8): 869-877.
[97] Choi D W, Jung J, Ha Y I, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites[J]. Plant Cell Reports, 2005, 23(8): 557-566.
[98] Kim M K, Lee B S, In J G, et al. Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf[J]. Plant Cell Reports, 2006, 25(6): 599-606.
[99] Phillips D R, Rasbery J M, Bartel B, et al. Biosynthetic diversity in plant triterpene cyclization[J]. Current Opinion in Plant Biology, 2006, 9(3): 305-314.
[100] Han J Y, Kwon Y S, Yang D C, et al. Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng[J]. Plant & Cell Physiology, 2006, 47(12): 1653-1662.
[101]罗志勇,刘水平,陆秋恒,等.人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析[J].生命科学研究, 2003, 7(04): 324-328.
[102]张崇禧,郑友兰,张春红,等.不同方法提取人参总皂苷工艺的优化研究[J].人参研究, 2003, 15(04): 5-8.
[103]张春红,张崇禧,郑友兰,等.浸渍法提取人参皂苷最佳工艺的研究[J].吉林农业大学学报, 2003, 25(01): 73-74.
[104]刘永练,郑成,毛桃嫣,等.微波提取人参中活性成分人参皂苷的研究[J].广州大学学报(自然科学版), 2006, 5(6): 52-55.
[105] Zhang S, Chen R, Wu H, et al. Ginsenoside extraction from Panax quinquefolium L. (American ginseng) root by using ultrahigh pressure[J]. J Pharm Biomed Anal, 2006, 41(1): 57-63.
[106]张晶,陈全成,弓晓杰,等.不同提取方法对人参皂苷提取率的影响[J].吉林农业大学学报, 2003, 25(01): 71-72.
[107]张宪臣,王淑敏,陈光,等.人参总皂苷超声提取工艺的研究[J].现代中药研究与实践, 2005, 19(06): 55-57.
[108]彭拓华,陈洁炜,黄湘兰,等.大孔树脂富集纯化人参总皂苷工艺条件优选[J].中药材, 2006, 29(04): 392-394.
[109]李洪刚,何克江,韩蔚,等.树脂联用纯化人参总皂苷[J].中医药学刊, 2005, 23(04): 708-711.
[110]桂双英,周亚球.比色法测定人参中人参总皂苷的含量[J].安徽中医学院学报, 2003, 22(04): 51-52.
[111]孔燕君,洪美凤.紫外分光光度法测定人参及三七中总皂苷含量[J].中国现代应用药学, 2000, 17(01): 51-52.
[112] Corthout J, Naessens T, Apers S, et al. Quantitative determination of ginsenosides from Panax ginseng roots and ginseng preparations by thin layer chromatography--densitometry[J]. J Pharm Biomed Anal, 1999, 21(1): 187-192.
[113]潘坚扬,程翼宇,王毅,等. 9种人参皂苷同时测定方法及在人参质量鉴别中的应用[J].分析化学, 2005, 33(11): 1565-1568.
[114] Chan T W, But P P, Cheng S W, et al. Differentiation and authentication of Panax ginseng, Panax quinquefolius, and ginseng products by using HPLC/MS[J]. Anal Chem, 2000, 72(6): 1281-1287.
[115] Fukuda N, Tanaka H, and Shoyama Y. Applications of ELISA, western blotting and immunoaffinity concentration for survey of ginsenosides in crude drugs of Panax species and traditional Chinese herbal medicines[J]. Analyst, 2000, 125(8): 1425-1429.
[116] Yoon S R, Nah J J, Kim S K, et al. Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay[J]. Chem Pharm Bull (Tokyo), 1998, 46(7): 1144-1147.
[117] Choi K, Kim M, Ryu J, et al. Ginsenosides compound K and Rh(2) inhibit tumor necrosis factor-alpha-induced activation of the NF-kappaB and JNK pathways in human astroglial cells[J]. Neurosci Lett, 2007, 421(1): 37-41.
[118] Bae E A, Han M J, Kim E J, et al. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants[J]. Arch Pharm Res, 2004, 27(1): 61-67.
[119] Cheng L Q, Kim M K, Lee J W, et al. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia[J]. Biotechnol Lett, 2006, 28(14): 1121-1127.
[120] Ko S R, Suzuki Y, Kim Y H, et al. Enzymatic synthesis of two ginsenoside Re-beta-xylosides[J]. Biosci Biotechnol Biochem, 2001, 65(5): 1223-1226.
[121] Ko S R, Suzuki Y, Choi K J, et al. Enzymatic preparation of genuine prosapogenin, 20(S)-ginsenoside Rh1, from ginsenosides Re and Rg1[J]. Biosci Biotechnol Biochem, 2000, 64(12): 2739-2743.
[122] Danieli B, Falcone L, Monti D, et al. Regioselective enzymatic glycosylation of natural polyhydroxylated compounds: galactosylation and glucosylation of protopanaxatriol ginsenosides[J]. J Org Chem, 2001, 66(1): 262-269.
[123] Wu J and Zhong J-J. Production of ginseng and its bioactive components in plant cell culture: Current technological and applied aspects[J]. Journal of Biotechnology, 1999, 68: 89-99.
[124] Lin L, Wu J, Ho K P, et al. Ultrasound-induced physiological effects and secondary metabolite (saponin) production in Panax ginseng cell cultures[J].Ultrasound Med Biol, 2001, 27(8): 1147-1152.
[125] Liu C J and Hou S S. Regulation of fungal elicitors on the cell growth and biosynthesis of saponin of Panax ginseng cell suspension culture[J]. Shi Yan Sheng Wu Xue Bao, 1999, 32(2): 168-174.
[126] Wu J and Ho K P. Assessment of various carbon sources and nutrient feeding strategies for Panax ginseng cell culture[J]. Appl Biochem Biotechnol, 1999, 82(1): 17-26.
[127] Thanh N T, Murthy H N, Yu K W, et al. Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng[J]. J Plant Physiol, 2006, 163(12): 1337-1341.
[128] Wu J Y, Wong K, Hoa K P, et al. Enhancement of saponin production in Panax ginseng cell culture by osmotic stress and nutrient feeding[J]. Enzyme and Microbial Technology, 2005, 36: 133-138.
[129] Yu K W, Murthy H N, Jeong C S, et al. Organic germanium stimulates the growth of ginseng adventitious roots and ginsenoside production [J]. Process Biochemistry 2005, 40: 2959–2961.
[130] Jeong C-S, Chakrabarty D, Hahna E-J, et al. Effects of oxygen, carbon dioxide and ethylene on growth and bioactive compound production in bioreactor culture of ginseng adventitious roots[J]. Biochemical Engineering Journal, 2006, 27: 252-263.
[131] Furuya T, Yoshikawa T, Ishii T, et al. Effects of auxins on growth and saponin production in callus cultures of Panax ginseng[J]. Planta Med, 1983, 47(3): 183-187.
[132] Furuya T, Yoshikawa T, Ishii T, et al. Regulation of saponin production in callus cultures of Panax ginseng[J]. Planta Med, 1983, 47(4): 200-204.
[133] Giri A and Narasu M L. Transgenic hairy roots: recent trends and applications[J]. Biotechnol Adv, 2000, 18(1): 1-22.
[134] Usami S, Morikawa S, Takebe I, et al. Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium[J]. Mol Gen Genet, 1987, 209(2): 221-226.
[135]张辉,于荣敏.发根农杆菌Ri质粒及其在植物次生代谢产物研究中的应用[J].沈阳药科大学学报, 2000, 17(04): 306-308.
[136] Hu Z-B and Du M. Hairy root and its application in plant genetic engineering[J]. Journal of Integrative Plant Biology 2006, 48(2): 121-127.
[137] Chilton M, Tepfer D, Petit A, et al. Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells.[J]. Nature, 1982, 295: 432-434.
[138] Ooms G, Twell D, Bossen M, et al. Development regulation of Ri T DNAgene expression in roots, shoots and tubers of transformed potato (Solanum tuberosum cv. Desiree)[J]. Plant Mol Biol, 1986, 6: 321-330.
[139] Stachel SE M E, Van Montagu M, Zambryski P. Identification of signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J]. Nature, 1985, 318: 624-629.
[140] Kumar V, Jones B, and Davey M. Transformation by Agrobacterium rhizogenes and regeneration of transgenic shoots of the wild soybean Glycine argyrea.[J]. Plant Cell Reports, 1991, 10: 135-138.
[141] Giri A, Banerjee S, Ahuja P, et al. Production of hairy roots in Aconitum heterophyllum wall using Agrobacterium rhizogenes[J]. In Vitro Cell Dev Biol-Plant, 1997, 33: 280-284.
[142] Nguyen C, Bourgaud F, Forlot P, et al. Establishment of hairy root cultures of Psoralea species[J]. Plant Cell Reports, 1992, 11: 424-427.
[143]张兴,刘晓娟,吕巧玲,等.毛状根生产次生代谢产物的研究进展[J].化工进展, 2007, 26(09): 1228-1232.
[144] Guillon S, Tremouillaux-Guiller J, Pati P K, et al. Harnessing the potential of hairy roots: dawn of a new era[J]. Trends Biotechnol, 2006, 24(9): 403-409.
[145] Sevon N, Drager B, Hiltunen R, et al. Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus[J]. Plant Cell Reports, 1997, 16: 605-611.
[146] Hoshino Y and Mii M. Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes[J]. Plant Cell Reports, 1998, 17: 256-261.
[147] Perez-Molphe E and Ochoa-Alejo N. Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes transformed tissues[J]. Plant Cell Reports, 1998, 17: 591-596.
[148] Handa T, Sujimura T, Kato E, et al. Genetic transformation of Eustoma grandiflorum with rol genes[J]. Acta Hort, 1995, 392: 209-218.
[149] Damiani F and Aricioni S. Transformation of Medicago arborea L. with Agrobacterium rhizogenes binary vector carrying the hygromycin resistance genes[J]. Plant Cell Reports, 1991(10): 300-303.
[150] Bourgaud F, Cravot A, Milesi S, et al. Production of plant secondary metabolites: a historical perspective[J]. Plant Science, 2001, 161: 839-851.
[151]张广求,王伯初,段传人,等.提高毛状根中次生代谢产物含量的方法与技术[J].重庆大学学报(自然科学版), 2005, 28(06): 121-124.
[152] Mateus L, Cherkaoui S, Christen P, et al. Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography[J].Phytochemistry, 2000, 54(5): 517-523.
[153] Pavlov A, Berkov S, Weber J, et al. Hyoscyamine biosynthesis in Datura stramonium hairy root in vitro systems with different ploidy levels[J]. Appl Biochem Biotechnol, 2008.
[154] Batra J, Dutta A, Singh D, et al. Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration[J]. Plant Cell Rep, 2004, 23(3): 148-154.
[155] Wu J Y, Ng J, Shi M, et al. Enhanced secondary metabolite (tanshinone) production of Salvia miltiorrhiza hairy roots in a novel root-bacteria coculture process[J]. Appl Microbiol Biotechnol, 2007, 77(3): 543-550.
[156] Morgan J A and Shanks J V. Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures[J]. J Biotechnol, 2000, 79(2): 137-145.
[157] Boitel-Conti M, Gontier E, Laberche J C, et al. Permeabilization of Datura innoxia hairy roots for release of stored tropane alkaloids[J]. Planta Med, 1995, 61(3): 287-290.
[158] Lee K, Yamakawa T, Kodama T, et al. Effects of chemicals on alkaloid production by transformed roots of Belladonna[J]. Phytochemistry, 1998, 49: 2343-2347.
[159] Yan Q, Hu Z, Tan R X, et al. Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation[J]. J Biotechnol, 2005, 119(4): 416-424.
[160] Rudrappa T, Neelwarne B, and Aswathanarayana R G.. In situ and Ex situ adsorption and recovery of betalains from hairy root cultures of Beta vulgaris[J]. Biotechnol Prog, 2004, 20(3): 777-785.
[161] Bais H P, Govindaswamy S, and Ravishankar G A. Enhancement of growth and coumarin production in hairy root cultures of witloof chicory (Cichorium intybus L.cv. Lucknow local) under the influence of fungal elicitors[J]. J Biosci Bioeng, 2000, 90(6): 648-653.
[162] Bhagwath S G and Hjortso M A. Statistical analysis of elicitation strategies for thiarubrine A production in hairy root cultures of Ambrosia artemisiifolia[J]. J Biotechnol, 2000, 80(2): 159-167.
[163] Chen H, Chena F, Chiu F C, et al. The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza[J]. Enzyme Microb Technol, 2001, 28(1): 100-105.
[164] Jeong G T, Park D H, Ryu H W, et al. Production of antioxidant compoundsby culture of Panax ginseng C.A. Meyer hairy roots: I. Enhanced production of secondary metabolite in hairy root cultures by elicitation[J]. Appl Biochem Biotechnol, 2005, 121-124: 1147-1157.
[165] Kim O T, Bang K H, Shin Y S, et al. Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate[J]. Plant Cell Rep, 2007, 26(11): 1941-1949.
[166] Komaraiah P, Reddy G V, Reddy P S, et al. Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum[J]. Biotechnol Lett, 2003, 25(8): 593-597.
[167] Pitta-Alvarez S I, Spollansky T C, and Giulietti A M. The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida[J]. Enzyme Microb Technol, 2000, 26(2-4): 252-258.
[168] Rijhwani S K and Shanks J V. Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures[J]. Biotechnol Prog, 1998, 14(3): 442-449.
[169] Magnotta M, Murata J, Chen J, et al. Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots[J]. Phytochemistry, 2007, 68(14): 1922-1931.
[170] Hughes E H, Hong S B, Gibson S I, et al. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine[J]. Metab Eng, 2004, 6(4): 268-276.
[171] Hong S B, Peebles C A, Shanks J V, et al. Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots[J]. J Biotechnol, 2006, 122(1): 28-38.
[172] Menzel G, Harloff H J, and Jung C. Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.)[J]. Appl Microbiol Biotechnol, 2003, 60(5): 571-576.
[173] Franks T K, Powell K S, Choimes S, et al. Consequences of transferring three sorghum genes for secondary metabolite (cyanogenic glucoside) biosynthesis to grapevine hairy roots[J]. Transgenic Res, 2006, 15(2): 181-195.
[174] Li X-Q, Tan A, Voegtline M, et al. Expression of Cry5B protein from Bacillus thuringiensis in plant roots confers resistance to root-knot nematode[J]. Biological Control, 2008, 47: 97–102.
[175] Gaume A, Komarnytsky S, Borisjuk N, et al. Rhizosecretion of recombinant proteins from plant hairy roots[J]. Plant Cell Rep, 2003, 21(12): 1188-1193.
[176] Shi H P and Lindemann P. Expression of recombinant Digitalis lanata EHRH. cardenolide 16'-O-glucohydrolase in Cucumis sativus L. hairy roots[J]. Plant Cell Rep, 2006, 25(11): 1193-1198.
[177] Kumar G B S, Ganapathi T R, Srinivas L, et al. Expression of hepatitis B surface antigen in potato hairy roots[J]. Plant Science, 2006, 170: 918–925.
[178] Jin U H, Chun J A, Lee J W, et al. Expression and characterization of extracellular fungal phytase in transformed sesame hairy root cultures[J]. Protein Expr Purif, 2004, 37(2): 486-492.
[179] Pletsch M, de Araujo B S, and Charlwood B V. Novel biotechnological approaches in environmental remediation research[J]. Biotechnol Adv, 1999, 17(8): 679-687.
[180] Gonz′alez P S, Capozucca C E, Tigierm H A, et al. Phytoremediation of phenol from wastewater, by peroxidases of tomato hairy root cultures[J]. Enzyme and Microbial Technology, 2006, 39: 647-653.
[181] Coniglio M S, Busto V D, Gonzalez P S, et al. Application of Brassica napus hairy root cultures for phenol removal from aqueous solutions[J]. Chemosphere, 2008, 72(7): 1035-1042.
[182] de Araujo B S, Dec J, Bollag J M, et al. Uptake and transformation of phenol and chlorophenols by hairy root cultures of Daucus carota, Ipomoea batatas and Solanum aviculare[J]. Chemosphere, 2006, 63(4): 642-651.
[183] Gonzalez P S, Agostini E, and Milrad S R. Comparison of the removal of 2,4-dichlorophenol and phenol from polluted water, by peroxidases from tomato hairy roots, and protective effect of polyethylene glycol[J]. Chemosphere, 2008, 70(6): 982-989.
[184] Rezek J, Macek T, Mackova M, et al. Plant metabolites of polychlorinated biphenyls in hairy root culture of black nightshade Solanum nigrum SNC-9O[J]. Chemosphere, 2007, 69(8): 1221-1227.
[185] Suresh B, Sherkhane P D, Kale S, et al. Uptake and degradation of DDT by hairy root cultures of Cichorium intybus and Brassica juncea[J]. Chemosphere, 2005, 61(9): 1288-1292.
[186] Nedelkoska T V and Doran P M. Hyperaccumulation of nickel by hairy roots of alyssum species: comparison with whole regenerated plants[J]. Biotechnol Prog, 2001, 17(4): 752-759.
[187] Nedelkoska T V and Doran P M. Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens[J]. Biotechnol Bioeng, 2000, 67(5): 607-615.
[188] Casas D A, Pitta-Alvarez S I, and Giulietti A M. Biotransformation ofhydroquinone by hairy roots of Brugmansia candida and effect of sugars and free-radical scavengers[J]. Appl Biochem Biotechnol, 1998, 69(2): 127-136.
[189] Yan C Y, Zhang Z, Yu R M, et al. Studies on biotransformation of arbutin by 4-hydroxy phenol in hairy root of Polygonum multiflorum[J]. Zhongguo Zhong Yao Za Zhi, 2007, 32(3): 192-195.
[190] Asada Y, Saito H, Yoshikawa T, et al. Biotransformation of 18 beta-glycyrrhetinic acid by ginseng hairy root culture[J]. Phytochemistry, 1993, 34(4): 1049-1052.
[191] Kanho H, Yaoya S, Kawahara N, et al. Biotransformation of benzaldehyde-type and acetophenone-type derivatives by Pharbitis nil hairy roots[J]. Chem Pharm Bull (Tokyo), 2005, 53(4): 361-365.
[192] Kwok K and Doran P. Kinetic and stoichiometric analysis of hairy roots in a segmented bubble column reactor[J]. Biotechnol Prog 1995, 11: 429-435.
[193]高文远,贾伟,段宏泉,等.药用植物发酵培养的工业化探讨[J].中国中药杂志, 2003, 28(05): 385-390.
[194] Kim Y, Wyslouzil B E, and Weathers P J. Secondary metabolism of hairy root cultures in bioreactors[J]. In Vitro Cell. Dev. Biol.-Plant, 2002, 38: 1-10.
[195] Hilton M G and Rhodes M J. Growth and hyoscyamine production of 'hairy root' cultures of Datura stramonium in a modified stirred tank reactor[J]. Appl Microbiol Biotechnol, 1990, 33(2): 132-138.
[196] Martin Y and Vermette P. Bioreactors for tissue mass culture: design, characterization, and recent advances[J]. Biomaterials, 2005, 26(35): 7481-7503.
[197]陈岺曦,王伯初.生物反应器与药用植物毛状根的大规模培养[J].生物技术通报, 2007(04): 38-41.
[198] Jeong G T, Park D H, Hwang B, et al. Studies on mass production of transformed Panax ginseng hairy roots in bioreactor[J]. Appl Biochem Biotechnol, 2002, 98-100: 1115-1127.
[199] Taya M, Yoyama A, Nomura R, et al. Production of peroxidase with horseradish hairy root cells in a two step culture system[J]. J Ferment Bioeng, 1989, 67: 31-34.
[200] Muranaka T, Ohakawa H, and Yamada Y. Scopolamine release into media by Duboisia leichhardtti hairy root clones[J]. Appl Microbiol Biotechnol, 1992, 37: 554-559.
[201] Kintzios S, Makri O, Pistola E, et al. Scale-up production of puerarin from hairy roots of Pueraria phaseoloides in an airlift bioreactor[J]. Biotechnol Lett, 2004, 26(13): 1057-1059.
[202] Peraza-Luna F, Rodriguez-Mendiola M, Arias-Castro C, et al. Sotoloneproduction by hairy root cultures of Trigonella foenum-graecum in airlift with mesh bioreactors[J]. J Agric Food Chem, 2001, 49(12): 6012-6019.
[203] Tescione L D, Ramakrishnan D, and Curtis W R. The role of liquid mixing and gas-phase dispersion in a submerged, sparged root reactor[J]. Enzyme Microb Technol, 1997, 20(3): 207-213.
[204] Pavlov A, Georgiev M, and Bley T. Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor[J]. Z Naturforsch [C], 2007, 62(5-6): 439-446.
[205] Souret F F, Kim Y, Wyslouzil B E, et al. Scale-up of Artemisia annua L. hairy root cultures produces complex patterns of terpenoid gene expression[J]. Biotechnol Bioeng, 2003, 83(6): 653-667.
[206] Woo S and Park J. Root culture using a mist culture system and estimation of scale-up feasibility[J]. J Chem Tech Biotechnol, 1996, 66: 355-362.
[207] Wyslouzil B E, Waterbury R G, and Weathers P J. The growth of single roots of Artemisia annua in nutrient mist reactors[J]. Biotechnol Bioeng, 2000, 70(2): 143-150.
[208] Kim, Kim Y, Wyslouzil, et al. A comparative study of mist and bubble column reactors in the in vitro production of artemisinin[J]. Plant Cell Reports, 2001, 20(5): 451-455.
[209] Uozumi N. Large-scale production of hairy root[J]. Adv Biochem Eng Biotechnol, 2004, 91: 75-103.
[210] Suresh B, Rajasekaran T, and Ramachandra S. Studies on osmolarity, conductivity and mass transfer for selection of a bioreactor for Tagetes patula L.hairy roots[J]. Process Biochemistry, 2001, 36(10): 987-993.
[211]唐正义,胡蓉.反义RNA研究进展[J].内江师范学院学报, 2004, 19(04): 30-35.
[212] Brantl S. Antisense-RNA regulation and RNA interference[J]. Biochim Biophys Acta, 2002, 1575(1-3): 15-25.
[213] Tomizawa J and Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA1 with primer transeript [J]. Proc Natl Acad Sci USA, 1981, 78: 1421-1425.
[214]娄虹,杨淑琴.反义RNA技术及其在农业领域的应用[J].辽宁大学学报(自然科学版), 2005, 32(04): 328-333.
[215]李伟明,张寒霜,赵俊丽,等.反义RNA技术及其在植物种质创新领域的应用.中国棉花学会2006年年会暨第七次代表大会论文汇编[C].保定: [出版者不祥],2006.
[216] Krol A and Mol J. Antisense genes in plants: an overview [J]. Gene, 1988, 72: 5-10.
[217] Oeller P, Iu M, and Tanlor P. Reversible inhibition of tomato fruit senescence by antisense RNA[J]. Science, 1991, 254: 437-439.
[218] Amemiya T, Kanayama Y, Yamaki S, et al. Fruit-specific V-ATPase suppression in antisense-transgenic tomato reduces fruit growth and seed formation[J]. Planta, 2006, 223(6): 1272-1280.
[219] Day A G, Bejarano E R, Buck K W, et al. Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus[J]. Proc Natl Acad Sci U S A, 1991, 88(15): 6721-6725.
[220] Bendahmane M and Gronenborn B. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA[J]. Plant Mol Biol, 1997, 33(2): 351-357.
[221] de Feyter R, Young M, Schroeder K, et al. A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco[J]. Mol Gen Genet, 1996, 250(3): 329-338.
[222] Levis C, Tronchet M, Meyer M, et al. Effects of antisense oligodeoxynucleotide hybridization on in vitro translation of potato virus Y RNA[J]. Virus Genes, 1992, 6(1): 33-46.
[223] Zhang P, Vanderschuren H, Futterer J, et al. Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes[J]. Plant Biotechnol J, 2005, 3(4): 385-397.
[224] Yui R, Iketani S, Mikami T, et al. Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility[J]. Plant J, 2003, 34(1): 57-66.
[225] Zhang Y, Shewry P R, Jones H, et al. Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley[J]. Plant J, 2001, 28(4): 431-441.
[226] Schmulling T, Rohrig H, Pilz S, et al. Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants[J]. Mol Gen Genet, 1993, 237(3): 385-394.
[227]张新宇,高燕宁. PCR引物设计及软件使用技巧[J].生物信息学, 2004(04): 1-8.
[228] Skrypina N A, Timofeeva A V, Khaspekov G L, et al. Total RNA suitable for molecular biology analysis[J]. J Biotechnol, 2003, 105(1-2): 1-9.
[229]王磊,范云六.植物微小RNA(microRNA)研究进展[J].中国农业科技导报, 2007, 9(3): 18-23.
[230]高文,贾洪涛.正义链还是反义链[J].临沂师范学院学报, 2003, 25(03): 62-63.
[231] Yoshikawa T and Furuya T. Saponin production by cultures of Panaxginseng transformed with Agrobacterium rhizogenes[J]. Plant Cell Reports, 1987, 6(6): 449-453.
[232]赵艳,于彦春.植物中转基因的整合机制[J].中国生物工程杂志, 2003, 23(2): 29-32.
[233] Shinozaki J, Shibuya M, Masuda K, et al. Squalene cyclase and oxidosqualene cyclase from a fern[J]. FEBS Lett, 2008, 582(2): 310-318.
[234]高瑜莹,裘爱泳,潘秋琴,等.植物甾醇的分析方法[J].中国油脂, 2001, 26(1): 25-28.
[235] Flores-Sanchez I, Ortega-Lopez J, Montes-Horcasitas M, et al. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.[J]. Plant Cell Physiol, 2002, 43: 1502-1509.
[236] Brian M, Carl A, Aideen H. Compositions with increased phytosterol levels obtained from plants with decreased triterpene saponin levels: US, 20070107081[P]. 2007-05-10.