高山红景天苯丙氨酸解氨酶基因的克隆及遗传转化
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摘要
高山红景天是一种非常具有开发前途的“适应原性”珍稀药用植物,其主要功效成分为红景天甙(salidroside)、甙元酪醇(tyrosol)等次生代谢产物。本文采用cDNA末端快速克隆技术(RACE),从高山红景天中首次分离得到苯丙烷类代谢途径中可能与酪醇生物合成相关的苯丙氨酸解氨酶(PAL)基因,命名为PALcll,对其基因特性、表达模式及PALcll基因差异表达组织中的红景天甙含量进行分析。在此基础上,构建了植物高效表达载体pBSPALcll并利用农杆菌介导法转化回高山红景天,经过分子鉴定证明PALcll基因已经整合到基因组DNA分子中并在转录水平大量表达。本研究为最终阐明甙元酪醇、红景天甙的生物合成代谢通路奠定了一定的基础。
Rhodiola sachalinensis (A.Bor), a kind of perennial herbal plant of Rhodiola genus, has been widely applied to many fields, such as foods, health protection and medicines. It's a prospecting rare and medicinal plant. Studies showes that it has remarkable functions on anti-oxygen deficiency, anti-radiation, anti-fatigue, anti-virus, anti-aging and so on. There are several factors leading to Rhodiola sachalinensis' low production, for example, demanding living environment, no special pollinating insect, low efficiency of pollination, reproductive barrier caused by physiological nature, and so on. The over utilization these years caused the destroy of it's ecology environment and it's natural resources was decreased sharply.Salidroside, and other secondary metabolites are main functional components of Rhodiola sachalinensis. Tyrosol is not only the effective and active monosomic of Rhodiola genus but also the direct substrate of Salidroside . At present, tyrosol synthesized in vivo can only be judged by its molecule structure and chemecial property. As tyrosol belongs to derivates of aromatic amino acid and is the typical simple phenolic compounds. Phen-propane' metabolic pathway is tyrosol's most probable biosynthetic pathway according to the commen rule of botan's secondary metabolic pathway. L-Phe ammom'a-lyase(PAL, EC 4.3.1.4) , which link primary metabolism and the first step of phen-propane' metabolism together, is a key enzyme and rate-limiting enzyme of this metabolic pathway and is a enzyme studied the most in phen-propane' metabolic pathway.A cDNA clone of PAL gene, named PALcll, which probably has some close relations to the biosynthetic of tyrosol was isolated from Rhodiola sachalinensis (GenBank accession number: AY879309), by RACE method. The sequence analysis
revealed that the lenth of PALcll cDNA is 2407bp, containing a complete open reading frame (ORF). It is predicted that PALcll encode a polypeptide of 77.45 kDa, consist of 710 amino acid residues, with its pi 6.50. There is a typical PAL structural domain between 55-586. Phylogenetic tree analysis showed that PALcll had a high sequence homology with other plant Phenylalanine ammonia-lyase, between 67%-82%. Studies on bioinformatics indicated that PALcll was a new member of PAL family. Studies on transmembrane domain indicated that there was no transmembrane domain in PALclP amino acid sequence and that PALcll was a typical matrix protein. Southern blot showed that there were many copies of PALcll gene in the genome of Sachalin rhodiola rhizome. Northern blot analysis showed that the PALcll was expressed more in callus than in leaves at transcriptional level at normal growth conditions. It's transcriptional level was improved after inducing by UV and was higher in callus than in leaves. Result of HPLC showed the correlation between transcription level and salidroside content in the callus and leaves was positively.On the basis above, to further investigate the function of PALcll, we constructed pBSPALcll and transformed it into Rhodiola sachalinensis via Agrobacterium. PCR, PCR-Southern and RT-PCR showed that the PALcll gene was integrated into Rhodiola sachalinensis genome and that its expression level in transgenic callus was much higher than that in wide type control and induced by UV.This study provides a solid foundation and theoretical basis for further investigation on the metabolism and biosynthesis of salidroside and tyrosol and offers some help for improving gennplasm resource of Rhodiola sachalinensis by genetic engineering means and raising the yields of salidroside and tyrosol.
Rhodiola sachalinensis (A.Bor), a kind of perennial herbal plant of Rhodiola genus, has been widely applied to many fields, such as foods, health protection and medicines. It's a prospecting rare and medicinal plant. Studies showes that it has remarkable functions on anti-oxygen deficiency, anti-radiation, anti-fatigue, anti-virus, anti-aging and so on. There are several factors leading to Rhodiola sachalinensis' low production, for example, demanding living environment, no special pollinating insect, low efficiency of pollination, reproductive barrier caused by physiological nature, and so on. The over utilization these years caused the destroy of it's ecology environment and it's natural resources was decreased sharply.Salidroside, and other secondary metabolites are main functional components of Rhodiola sachalinensis. Tyrosol is not only the effective and active monosomic of Rhodiola genus but also the direct substrate of Salidroside . At present, tyrosol synthesized in vivo can only be judged by its molecule structure and chemecial property. As tyrosol belongs to derivates of aromatic amino acid and is the typical simple phenolic compounds. Phen-propane' metabolic pathway is tyrosol's most probable biosynthetic pathway according to the commen rule of botan's secondary metabolic pathway. L-Phe ammom'a-lyase(PAL, EC 4.3.1.4) , which link primary metabolism and the first step of phen-propane' metabolism together, is a key enzyme and rate-limiting enzyme of this metabolic pathway and is a enzyme studied the most in phen-propane' metabolic pathway.A cDNA clone of PAL gene, named PALcll, which probably has some close relations to the biosynthetic of tyrosol was isolated from Rhodiola sachalinensis (GenBank accession number: AY879309), by RACE method. The sequence analysis
revealed that the lenth of PALcll cDNA is 2407bp, containing a complete open reading frame (ORF). It is predicted that PALcll encode a polypeptide of 77.45 kDa, consist of 710 amino acid residues, with its pi 6.50. There is a typical PAL structural domain between 55-586. Phylogenetic tree analysis showed that PALcll had a high sequence homology with other plant Phenylalanine ammonia-lyase, between 67%-82%. Studies on bioinformatics indicated that PALcll was a new member of PAL family. Studies on transmembrane domain indicated that there was no transmembrane domain in PALclP amino acid sequence and that PALcll was a typical matrix protein. Southern blot showed that there were many copies of PALcll gene in the genome of Sachalin rhodiola rhizome. Northern blot analysis showed that the PALcll was expressed more in callus than in leaves at transcriptional level at normal growth conditions. It's transcriptional level was improved after inducing by UV and was higher in callus than in leaves. Result of HPLC showed the correlation between transcription level and salidroside content in the callus and leaves was positively.On the basis above, to further investigate the function of PALcll, we constructed pBSPALcll and transformed it into Rhodiola sachalinensis via Agrobacterium. PCR, PCR-Southern and RT-PCR showed that the PALcll gene was integrated into Rhodiola sachalinensis genome and that its expression level in transgenic callus was much higher than that in wide type control and induced by UV.This study provides a solid foundation and theoretical basis for further investigation on the metabolism and biosynthesis of salidroside and tyrosol and offers some help for improving gennplasm resource of Rhodiola sachalinensis by genetic engineering means and raising the yields of salidroside and tyrosol.
引文
[1] 张彦福.新疆植物志.第二卷第二分册.乌鲁木齐:新疆科技卫生出版社,1995,242～243
[2] 新疆八一农学院.新疆植物检索表.第二册.乌鲁木齐:新疆人民出版社,1982,453～465
[3] 李伟.西藏红景天组织培养及红景天甙相关代谢酶的研究:[硕士学位论文].北京:首都师范大学,2003
[4] 宇陀·元丹贡布.四部医典.北京:人民卫生出版社,1985,54
[5] 明海泉,夏光成,张瑞钧,等.红景天研究进展.中草药,1988,19(5):37～42
[6] 蓝晓红,王莉,李艳丽.红景天的现代研究与进展.中国新医药,2003,2(2):27～28
[7] 白晓菊.高原人参—红景天.家庭医药,2004(9):16
[8] 贾国夫.红景天的研究概况及展望.四川草原,1998,3:38～39
[9] 高秋娜.红景天的药理与临床研究概述[J].河南中医,2000,2176～78
[10] 宋月英,韩慧文,郝素云,等.红景天研究进展.武警医院学报,2004(13):66～68
[11] 黄德昌,岳安云.高原人参—红景天生态气候条件浅析.气象,1994,20(3):241
[12] 南桂仙.高山红景天组织培养的研究:[硕士学位论文].延吉:延边大学,2004
[13] 罗明,谭敦炎,张玉霞,等.高山红景天的组织培养.新疆农业科学,1996(3):123～125
[14] 许建峰,刘传斌,方晓丹,等.高山红景天细胞悬浮培养中pH值对红景天甙胞外释放及细胞活性的影响.植物学报,1997,39(11):1022～1029
[15] 李有忠,孙和平,祁如虎,等.狭叶红景天营养器官的解剖结构及其生态学 意义.青海师范大学学报(自然科学版),1994,(4):34～40
[16] 安丰.四川红景天属植物的生药学研究.中国中药杂志,1993,18(8):456～459
[17] 王思宏,金香淑,姚艳红,等.野生和栽培高山红景天的核磁共振指纹图谱的研究.延边大学学报(自然科学版),2001,27(4):274～276
[18] 钱彦丛.红景天研究新进展.中药材,1995,18(10):499～501
[19] 胡挺松,陆一鸣,马兰青,等.红景天属4种植物RAPD分析与分类鉴定.中草药,2004,35(11):1286～1289
[20] 徐宝军,郑毅男,李向高,等.红景天属植物研究新进展.中药材,2000,23(9):580～584
[21] 胡挺松.红景天属4种药用植物RAPD分析及特异片段的克隆:[硕士学位论文].长春:中国人民解放军军需大学,2004
[22] 顾艳丽,王东凯,陈修毅,等.红景天研究进展.中国医药学报,2003,18(9):560～561
[23] 丁朝武.红景天苷和红景天苷元标准品的制备方法研究.分析测试学报,1997,16(1):38
[24] 刘声远.高山红景天根茎中超氧化歧化酶活性测定及种类鉴定.中草药,1993,24(3):125
[25] 孙晓军,赵慧,林杰.红景天的药理与制剂研究综述.中国药师,2005,8(5):415～416
[26] 赵文,等.藏产红景天混合提取物对BALB/c小鼠免疫功能的影响[J].实验动物科学与管理,1998,15(2):2～5
[27] 金永日,等.高山红景天茎叶提取物的初步药理研究[J].人参研究,2000,12(2):25～28
[28] 陈亚东,曹秀兰,田长有,等.高山红景天对小鼠耐缺氧、抗疲劳及耐低温作用的影响.中国中医药科技,2002,9(3):157～158
[29] 曹晓哲,邢传平,杨生岳,等.缺氧和营养不良对大鼠膈肌超微结构的影响 及红景天保护作用的电镜研究.高原医学杂志,1998,8(2):28～29
[30] 姜义,宵雪媛,关桂梅.红景天甙对x射线照射小鼠的预防作用.中华放射医学防护杂志,1995,15(3):214～216
[31] 明海泉.红景天甙的合成及药理作用.医学通报,1986(6):373～375
[32] 尹桂山,邹静琴,王桂香.红景天根乙醇浸液的营养延缓衰老作用研究.营养学报,1992,14(6):98～100
[33] 胡卡明,王尊凤,李立昆等.红景天胶囊治疗中医虚证100例临床报告.成都中医药大学学报,1998,21(2):17～19
[34] 崔旭.红景天复方制剂对家蝇寿命及脑组织自由基代谢的影响.白求恩医科大学学报,1995,21(4):347～350
[35] 孙非,于起福,孙寒.高山红景天多糖对病毒感染大鼠心肌细胞的抑制作用.中国药理学通报,1997,13(6):525～528
[36] 李伟,黄勤妮.红景天属植物的研究及应用.首都师范大学学报(自然科学版),2003,24(1):55～59
[37] 许建峰,应佩青,苏志国.高山红景天资源应用与开发研究进展.中草药,1998,29(3):202～205
[38] 许建峰,韩爱明,冯朴荪.高山红景天细胞悬浮培养生长和营养成分摄取动力学及其计量关系.应用与环境生物学报,1997,3(2):100～105
[39] 许建峰,苏志国.利用高山红景天培养细胞生物转化外源酪醇生产红景天甙的研究.植物学报,1998,40(12):1129～1135
[40] 戴勋.植物次生代谢.昭通师范高等专科学校学报,2002,24(5):35～38
[41] 马兰青.高山红景天红景天甙生物合成相关基因的克隆及功能分析:[博士学位论文].长春:吉林大学,2005
[42] Kessler A, Baldwin IT. Defensive function of herbivore-induced plant volatile emission in nature. Science, 2001, 291: 2141～2144
[43] 董妍玲,潘学武.植物次生代谢产物简介.生物学通报,2002,37(11):17～19
[44] 杨致荣,毛雪,李润植.植物次生代谢基因工程研究进展.植物生理与分子生物学学报,2005,31(1):11～18
[45] Winkel-Shirtey B. Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathway. Physiol Plant, 1999, 107: 142～149
[46] MeMullen MD, Byrne PF, Snook ME, et, al. Quantitative trait loci and metabolic pathways. Proc Nail Acad Sci USA, 1998, 95: 1996～2000
[47] 杜丽娜,张存莉,朱玮,等.植物次生代谢合成途径及生物学意义.西北林学院学报,2005,20(3):150～155
[48] 何水林.参与植物次生代谢调控的转录因子及其在植物次生代谢遗传改良中的应用.热带亚热带植物学报,2004,12(4):374～380
[49] 何水林,郑金贵,王晓峰,等.植物次生代谢:功能、调控及其基因工程.应用与环境生物学报,2002,8(5):558～563
[50] Dixon R. Natural products and plant disease resistance. Nature, 2001, 411(14): 843～847
[51] 吴双秀.高山红景天颗粒状愈伤组织悬浮培养与红景天甙的诱导:[博士学位论文].哈尔滨:东北林业大学,2001
[52] 许建峰.高山红景天愈伤组织颗粒悬浮培养生产红景天甙动力学与过程调控:[博士学位论文].大连:大连理工大学,1996
[53] Koukol J, Corm. E E. The metabolism of aromatic compounds in higher plants. Ⅳ. Purification and properties of the phenylalanine deaminase of Herdeum vulagare [J]. Journal of Biology and Chemistry, 1961, 236: 2692～2698
[54] 欧阳光察,应初衍.植物苯丙氨酸解氨酶的研究.Ⅳ.水稻、小麦PAL的纯化及基本特性[J].植物生理学报,1985,11(2):204～214
[55] JonesD H. Review Article Number 3. Phenylalanine ammonia-lyase: Regulation of its induction and its role in plant development. Phytochemisty, 1984, 23(7): 1349～1359
[56] Nakashima J. Immunocytochemical localization of phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase in differentiating tracheary elements derived from Zinnia mesop hyll cells. Plant Cell Physiology, 1997, 38: 113～123
[57] 欧阳光察,薛应龙.植物苯丙烷代谢的生理意义及调控[J].植物生理学通讯,1988,24(3):9～16
[58] Carom EL, Towers GHN. Review article: phenylanine ammonia lyase[J]. Phytochemisty, 1973, (12): 961～973
[59] Leyva A, Jafillo A, Salinas J, et al. Low temperature induces the accumulat ion of Phenylalanine ammonia-lyase and ehalcone synthasem RNA of Arabidopsis thaliana in a light-dependent manner. Plant Physiology, 1995, 108: 39～46
[60] Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Natl Acad Sci USA, 1995, 92: 5905～5909
[61] Kervinen T, Peltonen S, Teeri T H, et, al. Differential expression ofphenylalanine ammonia-lyase genes in barley induced by fungal infection and elicitors. New Phytol, 1998, 139: 293～300
[62] Mauch-Mani B, Slusarenko A J. Production of salicylic acid precursorsis amajor function of phenylalanine ammonia-lyase in the resistance of Arabidopsis to Peronospora parasitica. Plant Cell, 1996, 8: 203～212
[63] Lawtin M A. Elicitormodulation of pheylalanine in phaseolus vulgaris. Biochemistry and Biophysiology Acta, 1980, 633: 162～169
[64] Krause J,Reznik H. Investigation on flavol accumulation in Fagopyrum esculorrtum moench as influenced by Pand N deficiency I [J]. Pllan Zon Physiology. 1976. 79: 392～400
[65] 江昌俊,余有本.苯丙氨酸解氨酶研究进展(综述).安徽农业大学学报,2001,28(4):425～430
[66] 缪元颖,杨顺楷,刘成君.苯丙氨酸解氨酶的分子生物学研究进展.应用与环境生物学报,2002,8(6):672～675
[67] Tanaka Y. Structure and characterization of a cDNA for phenylalanine ammonia-lyase from cutinjured roots of sweet potato. Plant Physiology, 1989, 90: 1403～1407
[68] Ohl S. Functional properties of a phenylalanine arnmonia-lyase promoter from Arabidopsis. Plant Cell, 1990, 2: 837～848
[69] Shinji Kawamata. Temporal and spatial pattern of expression of the pea phenylalanine ammonia-lyase gene promoter in transgenic tobacco. Plant Cell Physiology, 1997, 38 (7): 792～803
[70] Lois R, Dietrich A, Hahlbrock K. A phenylalanine ammonia-lyase gene from Parsley: Structure, regulation and identification of elicitor and light responsive-cis-acting elements. EMBOJ, 1989, 8: 1641～1648
[71] Whetten R W, Sederoff R R. Phenylalanine ammonia-lyase from loblolly pine: purification of the enzyme and isolation of complementary DNA clones. Plant Physiology, 1992, 98: 380～386
[72] Minami E. Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants. Europe Journal of Biochemistry, 1989, 185: 18～25
[73] Nagai N, Kitauchi F, Shimosaka M, et al. Cloning and sequencing of a full-lengthcDNA coding from phenylalanine ammonia-lyase from tobacco cell culture. Plant Physiology, 1994, 104: 1091～1092
[74] Lee SW. Truncated phenylalanine ammonia-lyase expression in tomato(Lyco persicon esculentnm). Journal of Biology and Chemistry,1999, 267: 11824～11830
[75] Cramerm C L, Edwards K. Phenylalanine ammonia-lyase gene organization and structure. Plant Molecular Biology, 1989 (12): 367
[76] Diallinas G, Kanellis A K. A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding. Plant Molecular Biology, 1994, 26(1): 473～479
[77] Goeri G. Stress responses in alfalfa (Medicago sativa L.). Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants. Plant Molecular Biology, 1991, 17(3): 415～429
[78] Matsumoto S. Molecular cloning of phenylalanine ammonia-lyase cDNA and classifica (B-66 ion of varieties and cultivars of tea plants (Camellia sininsis) using the rice PAL cDNA probe. Theoretical and Applied Genetics, 1994, 89 (6): 671～675
[79] 金雪丽,吴松权,石铁源,等.高山红景天花型调查.延边大学农学学报,2001,23(2):138～139
[80] 迪芬巴赫C W著.PCR技术实验指南.黄培堂译.科学出版社.2000:234～236
[81] 彭江南,马成禹,葛永潮.大花红景天的化学成分.中草药,1995,26(4):177～179
[82] 陈玉婷,谷燕莉,李佳.HPLC测定红景天药材主流品种及同属5种植物的红景天苷含量.北京中医药大学学报,2003,26:48～51
[83] 李和平,廖玉才.两个紧密连锁的小麦苯丙氨酸解氨酶基因的分离与鉴定.遗传学报,2003,30:907～912
[84] 余沛涛,薛应龙.植物苯丙氨酸解氨酶(PAL)在细胞分化中的作用.植物生理学报,1987,131:14～19
[85] Rubery R H, Northcote D H. Site of phenylalanine ammonia-lyase activity and synthesis of lignin during xylem differention. Nature (London), 1968, 210: 1230～1234
[86] Landtaga J, Baumertb A, Degenkolbc T, et al. Accumulation of tyrosol glu-coside in transgenic potato plants expressing a parsley tyrosine decarboxylase. Phytochemistry, 2002: 683～689
[87] 布坎南B B,格鲁依森姆W,琼斯R L.植物生物化学与分子生物学.瞿礼 嘉,顾红雅,白书农,等主译.科学出版社。2004,1052～1060
[88] Hrazdina G. Compartmentation in aromatic metabolism. In: Ibrahim RK. Recent Advances in Phytochemistry. New York: Plenum Press, 1992: 1～33
[89] 刘传斌,金郁,李宁,等.不同来源高山红景天材料中有效成分的HPLC分析.天然产物研究与开发,1997,11:18～22
[2] 新疆八一农学院.新疆植物检索表.第二册.乌鲁木齐:新疆人民出版社,1982,453～465
[3] 李伟.西藏红景天组织培养及红景天甙相关代谢酶的研究:[硕士学位论文].北京:首都师范大学,2003
[4] 宇陀·元丹贡布.四部医典.北京:人民卫生出版社,1985,54
[5] 明海泉,夏光成,张瑞钧,等.红景天研究进展.中草药,1988,19(5):37～42
[6] 蓝晓红,王莉,李艳丽.红景天的现代研究与进展.中国新医药,2003,2(2):27～28
[7] 白晓菊.高原人参—红景天.家庭医药,2004(9):16
[8] 贾国夫.红景天的研究概况及展望.四川草原,1998,3:38～39
[9] 高秋娜.红景天的药理与临床研究概述[J].河南中医,2000,2176～78
[10] 宋月英,韩慧文,郝素云,等.红景天研究进展.武警医院学报,2004(13):66～68
[11] 黄德昌,岳安云.高原人参—红景天生态气候条件浅析.气象,1994,20(3):241
[12] 南桂仙.高山红景天组织培养的研究:[硕士学位论文].延吉:延边大学,2004
[13] 罗明,谭敦炎,张玉霞,等.高山红景天的组织培养.新疆农业科学,1996(3):123～125
[14] 许建峰,刘传斌,方晓丹,等.高山红景天细胞悬浮培养中pH值对红景天甙胞外释放及细胞活性的影响.植物学报,1997,39(11):1022～1029
[15] 李有忠,孙和平,祁如虎,等.狭叶红景天营养器官的解剖结构及其生态学 意义.青海师范大学学报(自然科学版),1994,(4):34～40
[16] 安丰.四川红景天属植物的生药学研究.中国中药杂志,1993,18(8):456～459
[17] 王思宏,金香淑,姚艳红,等.野生和栽培高山红景天的核磁共振指纹图谱的研究.延边大学学报(自然科学版),2001,27(4):274～276
[18] 钱彦丛.红景天研究新进展.中药材,1995,18(10):499～501
[19] 胡挺松,陆一鸣,马兰青,等.红景天属4种植物RAPD分析与分类鉴定.中草药,2004,35(11):1286～1289
[20] 徐宝军,郑毅男,李向高,等.红景天属植物研究新进展.中药材,2000,23(9):580～584
[21] 胡挺松.红景天属4种药用植物RAPD分析及特异片段的克隆:[硕士学位论文].长春:中国人民解放军军需大学,2004
[22] 顾艳丽,王东凯,陈修毅,等.红景天研究进展.中国医药学报,2003,18(9):560～561
[23] 丁朝武.红景天苷和红景天苷元标准品的制备方法研究.分析测试学报,1997,16(1):38
[24] 刘声远.高山红景天根茎中超氧化歧化酶活性测定及种类鉴定.中草药,1993,24(3):125
[25] 孙晓军,赵慧,林杰.红景天的药理与制剂研究综述.中国药师,2005,8(5):415～416
[26] 赵文,等.藏产红景天混合提取物对BALB/c小鼠免疫功能的影响[J].实验动物科学与管理,1998,15(2):2～5
[27] 金永日,等.高山红景天茎叶提取物的初步药理研究[J].人参研究,2000,12(2):25～28
[28] 陈亚东,曹秀兰,田长有,等.高山红景天对小鼠耐缺氧、抗疲劳及耐低温作用的影响.中国中医药科技,2002,9(3):157～158
[29] 曹晓哲,邢传平,杨生岳,等.缺氧和营养不良对大鼠膈肌超微结构的影响 及红景天保护作用的电镜研究.高原医学杂志,1998,8(2):28～29
[30] 姜义,宵雪媛,关桂梅.红景天甙对x射线照射小鼠的预防作用.中华放射医学防护杂志,1995,15(3):214～216
[31] 明海泉.红景天甙的合成及药理作用.医学通报,1986(6):373～375
[32] 尹桂山,邹静琴,王桂香.红景天根乙醇浸液的营养延缓衰老作用研究.营养学报,1992,14(6):98～100
[33] 胡卡明,王尊凤,李立昆等.红景天胶囊治疗中医虚证100例临床报告.成都中医药大学学报,1998,21(2):17～19
[34] 崔旭.红景天复方制剂对家蝇寿命及脑组织自由基代谢的影响.白求恩医科大学学报,1995,21(4):347～350
[35] 孙非,于起福,孙寒.高山红景天多糖对病毒感染大鼠心肌细胞的抑制作用.中国药理学通报,1997,13(6):525～528
[36] 李伟,黄勤妮.红景天属植物的研究及应用.首都师范大学学报(自然科学版),2003,24(1):55～59
[37] 许建峰,应佩青,苏志国.高山红景天资源应用与开发研究进展.中草药,1998,29(3):202～205
[38] 许建峰,韩爱明,冯朴荪.高山红景天细胞悬浮培养生长和营养成分摄取动力学及其计量关系.应用与环境生物学报,1997,3(2):100～105
[39] 许建峰,苏志国.利用高山红景天培养细胞生物转化外源酪醇生产红景天甙的研究.植物学报,1998,40(12):1129～1135
[40] 戴勋.植物次生代谢.昭通师范高等专科学校学报,2002,24(5):35～38
[41] 马兰青.高山红景天红景天甙生物合成相关基因的克隆及功能分析:[博士学位论文].长春:吉林大学,2005
[42] Kessler A, Baldwin IT. Defensive function of herbivore-induced plant volatile emission in nature. Science, 2001, 291: 2141～2144
[43] 董妍玲,潘学武.植物次生代谢产物简介.生物学通报,2002,37(11):17～19
[44] 杨致荣,毛雪,李润植.植物次生代谢基因工程研究进展.植物生理与分子生物学学报,2005,31(1):11～18
[45] Winkel-Shirtey B. Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathway. Physiol Plant, 1999, 107: 142～149
[46] MeMullen MD, Byrne PF, Snook ME, et, al. Quantitative trait loci and metabolic pathways. Proc Nail Acad Sci USA, 1998, 95: 1996～2000
[47] 杜丽娜,张存莉,朱玮,等.植物次生代谢合成途径及生物学意义.西北林学院学报,2005,20(3):150～155
[48] 何水林.参与植物次生代谢调控的转录因子及其在植物次生代谢遗传改良中的应用.热带亚热带植物学报,2004,12(4):374～380
[49] 何水林,郑金贵,王晓峰,等.植物次生代谢:功能、调控及其基因工程.应用与环境生物学报,2002,8(5):558～563
[50] Dixon R. Natural products and plant disease resistance. Nature, 2001, 411(14): 843～847
[51] 吴双秀.高山红景天颗粒状愈伤组织悬浮培养与红景天甙的诱导:[博士学位论文].哈尔滨:东北林业大学,2001
[52] 许建峰.高山红景天愈伤组织颗粒悬浮培养生产红景天甙动力学与过程调控:[博士学位论文].大连:大连理工大学,1996
[53] Koukol J, Corm. E E. The metabolism of aromatic compounds in higher plants. Ⅳ. Purification and properties of the phenylalanine deaminase of Herdeum vulagare [J]. Journal of Biology and Chemistry, 1961, 236: 2692～2698
[54] 欧阳光察,应初衍.植物苯丙氨酸解氨酶的研究.Ⅳ.水稻、小麦PAL的纯化及基本特性[J].植物生理学报,1985,11(2):204～214
[55] JonesD H. Review Article Number 3. Phenylalanine ammonia-lyase: Regulation of its induction and its role in plant development. Phytochemisty, 1984, 23(7): 1349～1359
[56] Nakashima J. Immunocytochemical localization of phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase in differentiating tracheary elements derived from Zinnia mesop hyll cells. Plant Cell Physiology, 1997, 38: 113～123
[57] 欧阳光察,薛应龙.植物苯丙烷代谢的生理意义及调控[J].植物生理学通讯,1988,24(3):9～16
[58] Carom EL, Towers GHN. Review article: phenylanine ammonia lyase[J]. Phytochemisty, 1973, (12): 961～973
[59] Leyva A, Jafillo A, Salinas J, et al. Low temperature induces the accumulat ion of Phenylalanine ammonia-lyase and ehalcone synthasem RNA of Arabidopsis thaliana in a light-dependent manner. Plant Physiology, 1995, 108: 39～46
[60] Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Natl Acad Sci USA, 1995, 92: 5905～5909
[61] Kervinen T, Peltonen S, Teeri T H, et, al. Differential expression ofphenylalanine ammonia-lyase genes in barley induced by fungal infection and elicitors. New Phytol, 1998, 139: 293～300
[62] Mauch-Mani B, Slusarenko A J. Production of salicylic acid precursorsis amajor function of phenylalanine ammonia-lyase in the resistance of Arabidopsis to Peronospora parasitica. Plant Cell, 1996, 8: 203～212
[63] Lawtin M A. Elicitormodulation of pheylalanine in phaseolus vulgaris. Biochemistry and Biophysiology Acta, 1980, 633: 162～169
[64] Krause J,Reznik H. Investigation on flavol accumulation in Fagopyrum esculorrtum moench as influenced by Pand N deficiency I [J]. Pllan Zon Physiology. 1976. 79: 392～400
[65] 江昌俊,余有本.苯丙氨酸解氨酶研究进展(综述).安徽农业大学学报,2001,28(4):425～430
[66] 缪元颖,杨顺楷,刘成君.苯丙氨酸解氨酶的分子生物学研究进展.应用与环境生物学报,2002,8(6):672～675
[67] Tanaka Y. Structure and characterization of a cDNA for phenylalanine ammonia-lyase from cutinjured roots of sweet potato. Plant Physiology, 1989, 90: 1403～1407
[68] Ohl S. Functional properties of a phenylalanine arnmonia-lyase promoter from Arabidopsis. Plant Cell, 1990, 2: 837～848
[69] Shinji Kawamata. Temporal and spatial pattern of expression of the pea phenylalanine ammonia-lyase gene promoter in transgenic tobacco. Plant Cell Physiology, 1997, 38 (7): 792～803
[70] Lois R, Dietrich A, Hahlbrock K. A phenylalanine ammonia-lyase gene from Parsley: Structure, regulation and identification of elicitor and light responsive-cis-acting elements. EMBOJ, 1989, 8: 1641～1648
[71] Whetten R W, Sederoff R R. Phenylalanine ammonia-lyase from loblolly pine: purification of the enzyme and isolation of complementary DNA clones. Plant Physiology, 1992, 98: 380～386
[72] Minami E. Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants. Europe Journal of Biochemistry, 1989, 185: 18～25
[73] Nagai N, Kitauchi F, Shimosaka M, et al. Cloning and sequencing of a full-lengthcDNA coding from phenylalanine ammonia-lyase from tobacco cell culture. Plant Physiology, 1994, 104: 1091～1092
[74] Lee SW. Truncated phenylalanine ammonia-lyase expression in tomato(Lyco persicon esculentnm). Journal of Biology and Chemistry,1999, 267: 11824～11830
[75] Cramerm C L, Edwards K. Phenylalanine ammonia-lyase gene organization and structure. Plant Molecular Biology, 1989 (12): 367
[76] Diallinas G, Kanellis A K. A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding. Plant Molecular Biology, 1994, 26(1): 473～479
[77] Goeri G. Stress responses in alfalfa (Medicago sativa L.). Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants. Plant Molecular Biology, 1991, 17(3): 415～429
[78] Matsumoto S. Molecular cloning of phenylalanine ammonia-lyase cDNA and classifica (B-66 ion of varieties and cultivars of tea plants (Camellia sininsis) using the rice PAL cDNA probe. Theoretical and Applied Genetics, 1994, 89 (6): 671～675
[79] 金雪丽,吴松权,石铁源,等.高山红景天花型调查.延边大学农学学报,2001,23(2):138～139
[80] 迪芬巴赫C W著.PCR技术实验指南.黄培堂译.科学出版社.2000:234～236
[81] 彭江南,马成禹,葛永潮.大花红景天的化学成分.中草药,1995,26(4):177～179
[82] 陈玉婷,谷燕莉,李佳.HPLC测定红景天药材主流品种及同属5种植物的红景天苷含量.北京中医药大学学报,2003,26:48～51
[83] 李和平,廖玉才.两个紧密连锁的小麦苯丙氨酸解氨酶基因的分离与鉴定.遗传学报,2003,30:907～912
[84] 余沛涛,薛应龙.植物苯丙氨酸解氨酶(PAL)在细胞分化中的作用.植物生理学报,1987,131:14～19
[85] Rubery R H, Northcote D H. Site of phenylalanine ammonia-lyase activity and synthesis of lignin during xylem differention. Nature (London), 1968, 210: 1230～1234
[86] Landtaga J, Baumertb A, Degenkolbc T, et al. Accumulation of tyrosol glu-coside in transgenic potato plants expressing a parsley tyrosine decarboxylase. Phytochemistry, 2002: 683～689
[87] 布坎南B B,格鲁依森姆W,琼斯R L.植物生物化学与分子生物学.瞿礼 嘉,顾红雅,白书农,等主译.科学出版社。2004,1052～1060
[88] Hrazdina G. Compartmentation in aromatic metabolism. In: Ibrahim RK. Recent Advances in Phytochemistry. New York: Plenum Press, 1992: 1～33
[89] 刘传斌,金郁,李宁,等.不同来源高山红景天材料中有效成分的HPLC分析.天然产物研究与开发,1997,11:18～22