西藏红景天组织培养及红景天甙相关代谢酶的研究
|
摘要
珍贵藏药植物——西藏红景天的根中含有以红景天甙为主的次生代谢物质，具有抗疲劳、抗衰老、抗缺氧、抗紫外线辐射等作用，是非常有开发前途的环境适应性药用濒危植物。本实验利用植物组织培养技术，筛选出红景天诱导愈伤组织、生芽、生根的合适激素配比的培养基，并探索了红景天甙可能代谢途径中几种调控酶的活性，以及不同外植体来源的愈伤组织在不同培养温度条件下不同继代培养中其酶活性与红景天甙含量的关系。
我们发现：(1)MS+6BA 2.0mgL~(-1)+NAA0.2mgL~(-1)、MS+6BA 3.0mgL~(-1)+NAA0.3mgL~(-1)和MS+6BA 2.0mgL~(-1)+IAA0.25mgL~(-1)都能较快地诱导出愈伤组织；狭叶红景天和大花红景天的叶片比茎较易产生愈伤组织，而且两者生成的愈伤组织的状态差别较大；在6-BA与NAA浓度之比等于10时，才能诱导出较好的愈伤组织，在IAA和GA共同存在的条件下则利于诱导芽的生长；MS和B5均+30gL~(-1)蔗糖+8gL~(-1)琼脂+IAA0.5mgL~(-1)(光照度8h d~(-1)、pH5.8)诱导生根最佳，两者的区别在于B5诱导出的根比MS所诱导的要粗，但较短。MS所诱导的根比B5所诱导的根要长。(2)、愈伤组织的外植体来源、培养温度条件和不同的继代培养数都影响着其中红景天甙的含量和苯丙氨酸解氨酶(PAL)、肉桂酸解氨酶(CA4H)和酪氨酸解氨酶(TAL)这3种酶的活性；不管是叶来源还是茎来源，不管是24℃培养还是4℃培养的愈伤组织，在连续继代培养中红景天甙含量的变化趋势基本上是一致的；无论是叶来源的愈伤组织还是茎来源的愈伤组织，培养温度对PAL酶的酶活性影响最大，对CA4H酶的酶活性影响最小，对TAL酶的酶活性影响视不同的外植体来源而定；在相同外植体来源的愈伤组织中及不同的培养温度的条件下，或是在不同外植体来源的愈伤组织中及相同温度的培养条件下，其红景天甙含量与PAL酶、CA4H酶和TAL酶的酶活性之间没有完全一致的对应伴随关系。推测可能在不同的组织器官中和不同温度条件下，红景天甙存在不同的占主导地位的代谢途径。
我们还根据苯丙氨酸解氨酶(PAL)基因家族的保守序列，以大花红景天DNA为模板，通过PCR扩增得到其约950bp长的PAL基因的克隆，测序并比较了它与其基因家族的同源性。
The Tibetan folk medical plant Rhodiola rosea was usually planted in a special environment in alpine plateau areas, such roots of which are used to cure senility,fatigue in man, and also to improve human immunity so as to be recovered from many diseases significantly. In this paper,we studied MS medium was used for the callus and bud, and MS or B5 medium were used for the root induction of both the leave and the stem of the Rhodiola rosea. MS and B5 medium supplemented with different ration of hormone and concentration sucrose and agrose. The Salidroside is the one of the most pharmacological activity-qualitity matters in Rhodiola rosea, and the determination of which in the callus of Rhodiola Kirilowir was made by HPLC .Then we studied the activity of enzymes PAL ,CA4H and TAL which may be the important enzymes in the metabolic pathway of Salidroside in the callus of Rhodiola Kirilowir. The results showed that:
(l)The best mediums to the callus of the Rhodiola rosea were MS+6BA 2.0mg L-1+NAA0.2mg L-1,MS+6BA 3.0mg L-1+NAA0.3mg L-1,MS+6BA 2.0mg L-1 +IAA0.25mg L-1. And it was easier to the induction of the callus for leave than stem ,the IAA and GA was extremely remarkable to the bud growth.The best medium to the root induction were MS and B5+30g L-1 sucrose+8g L-1agrose+IAA 0.5mg L -1 with the light of 8 h d-1 and pH5.8,the difference between the two mediums was that the root of the former was thinner and longer than the latter.
(2)The explant ,the temperature and the growth era of the callus for Rhodiola Kirilowir all worked on both the content of the Salidroside and the activity of enzymes including PAL ,CA4H and TAL in the callus. With 24 C or 4 C, the change trends of the content of the Salidroside was basically consentaneous in the consecutive cultural eras of the callus. In the callus from the different explants,the influence was maximal to the the activity of PAL enzyme and the influence was lowest to the the activity of CA4H enzyme, and the influence to the the activity of TAL enzyme was ascertained according to the explant of the callus. So in the same explant with the different temperature or in the different explant with the same temperature, there was no incident between the content of the Salidroside and the activity of enzymes PAL ,CA4H and TAL, and We presumed that there may be emphasized particularly on different metabolic pathway of Salidroside. According to the conserved domain of the PAL gene family, we achieved to the segment of PAL gene about 950bp in Rhodiola crenulata .Some proposals were put forward for further development of the important enzymes in the metabolic pathway of Salidroside.
我们发现：(1)MS+6BA 2.0mgL~(-1)+NAA0.2mgL~(-1)、MS+6BA 3.0mgL~(-1)+NAA0.3mgL~(-1)和MS+6BA 2.0mgL~(-1)+IAA0.25mgL~(-1)都能较快地诱导出愈伤组织；狭叶红景天和大花红景天的叶片比茎较易产生愈伤组织，而且两者生成的愈伤组织的状态差别较大；在6-BA与NAA浓度之比等于10时，才能诱导出较好的愈伤组织，在IAA和GA共同存在的条件下则利于诱导芽的生长；MS和B5均+30gL~(-1)蔗糖+8gL~(-1)琼脂+IAA0.5mgL~(-1)(光照度8h d~(-1)、pH5.8)诱导生根最佳，两者的区别在于B5诱导出的根比MS所诱导的要粗，但较短。MS所诱导的根比B5所诱导的根要长。(2)、愈伤组织的外植体来源、培养温度条件和不同的继代培养数都影响着其中红景天甙的含量和苯丙氨酸解氨酶(PAL)、肉桂酸解氨酶(CA4H)和酪氨酸解氨酶(TAL)这3种酶的活性；不管是叶来源还是茎来源，不管是24℃培养还是4℃培养的愈伤组织，在连续继代培养中红景天甙含量的变化趋势基本上是一致的；无论是叶来源的愈伤组织还是茎来源的愈伤组织，培养温度对PAL酶的酶活性影响最大，对CA4H酶的酶活性影响最小，对TAL酶的酶活性影响视不同的外植体来源而定；在相同外植体来源的愈伤组织中及不同的培养温度的条件下，或是在不同外植体来源的愈伤组织中及相同温度的培养条件下，其红景天甙含量与PAL酶、CA4H酶和TAL酶的酶活性之间没有完全一致的对应伴随关系。推测可能在不同的组织器官中和不同温度条件下，红景天甙存在不同的占主导地位的代谢途径。
我们还根据苯丙氨酸解氨酶(PAL)基因家族的保守序列，以大花红景天DNA为模板，通过PCR扩增得到其约950bp长的PAL基因的克隆，测序并比较了它与其基因家族的同源性。
The Tibetan folk medical plant Rhodiola rosea was usually planted in a special environment in alpine plateau areas, such roots of which are used to cure senility,fatigue in man, and also to improve human immunity so as to be recovered from many diseases significantly. In this paper,we studied MS medium was used for the callus and bud, and MS or B5 medium were used for the root induction of both the leave and the stem of the Rhodiola rosea. MS and B5 medium supplemented with different ration of hormone and concentration sucrose and agrose. The Salidroside is the one of the most pharmacological activity-qualitity matters in Rhodiola rosea, and the determination of which in the callus of Rhodiola Kirilowir was made by HPLC .Then we studied the activity of enzymes PAL ,CA4H and TAL which may be the important enzymes in the metabolic pathway of Salidroside in the callus of Rhodiola Kirilowir. The results showed that:
(l)The best mediums to the callus of the Rhodiola rosea were MS+6BA 2.0mg L-1+NAA0.2mg L-1,MS+6BA 3.0mg L-1+NAA0.3mg L-1,MS+6BA 2.0mg L-1 +IAA0.25mg L-1. And it was easier to the induction of the callus for leave than stem ,the IAA and GA was extremely remarkable to the bud growth.The best medium to the root induction were MS and B5+30g L-1 sucrose+8g L-1agrose+IAA 0.5mg L -1 with the light of 8 h d-1 and pH5.8,the difference between the two mediums was that the root of the former was thinner and longer than the latter.
(2)The explant ,the temperature and the growth era of the callus for Rhodiola Kirilowir all worked on both the content of the Salidroside and the activity of enzymes including PAL ,CA4H and TAL in the callus. With 24 C or 4 C, the change trends of the content of the Salidroside was basically consentaneous in the consecutive cultural eras of the callus. In the callus from the different explants,the influence was maximal to the the activity of PAL enzyme and the influence was lowest to the the activity of CA4H enzyme, and the influence to the the activity of TAL enzyme was ascertained according to the explant of the callus. So in the same explant with the different temperature or in the different explant with the same temperature, there was no incident between the content of the Salidroside and the activity of enzymes PAL ,CA4H and TAL, and We presumed that there may be emphasized particularly on different metabolic pathway of Salidroside. According to the conserved domain of the PAL gene family, we achieved to the segment of PAL gene about 950bp in Rhodiola crenulata .Some proposals were put forward for further development of the important enzymes in the metabolic pathway of Salidroside.
引文
[1] (美)J．萨姆布鲁克 E F.T.曼尼阿蒂斯 分子克隆实验指南(第二版)，科学出版社，1992．
[2] 陈敏，廖志华等．生物技术在长春花研究中的应用．中草药，2001，32(1)：78—81．
[3] 陈书安，王晓东等．藏红花的研究进展．中草药，2001，32(12)：1137—1139．
[4] 陈晓亚，刘培等．植物次生代谢的分子生物学及基因工程．生命科学，1996，8(2)：8—10．
[5] 陈晓亚，刘培等．植物的次生代谢．科学，1996，48(5)：37—39．
[6] 成静，郭勇．植物细胞工程药物生产的研究进展．江西科学，2000，18(1)：60—62．
[7] 崔堂兵，郭勇等．植物组织培养中褐变现象的产生机理及克服方法．广东农业科学，2001，3：16—18．
[8] 戴良先，川西高原红景天产业化开发的思考．四川草原，2000，2：61—64．
[9] 高国训．植物组织培养中的褐变问题．植物生理学通讯，1999，35(6)：501—506．
[10] 韩爱明．许建峰．方晓丹等．影响高山红景天细胞悬浮培养中细胞生长和红景天甙积累的几个因素．植物生理学通讯1997,33(1)：33-36．
[11] 胡国武，温廷益等．紫杉醇合成代谢途径中紫杉烯合成酶cDNA的克隆．生物工程学报，2000，16(2)：157—159．
[12] 胡维，刘敬忠等．苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达及酶法合成L-苯丙氨酸．微生物学杂志，2000，20(3)：30—32．
[13] 贾冬梅，李春荣等．高山红景天开发概述与应用开发建议．黑河科技，2000，4：6—7．
[14] 贾国夫．红景天的研究概况及展望．四川草原，1997，3：38—40．
[15] 江昌俊，余有本．苯丙氨酸解氨酶的研究进展．安徽农业大学学报，2001，28(4)：425—430．
[16] 姜明兰．钟文田．韩洪等．组织培养生产高山红景天有效成分的研究Ⅰ．高山红景天愈伤组织的诱导与培养．沈阳农业大学学报，1994,25(4)：355—359．
[17] 李永明，赵玉琪等．实用分子生物学方法手册，科学出版社．1998．
[18] 刘春朝，王玉春等．生物诱导子调节植物组织次生代谢的研究．植物学通报，1999，16(2)131—137．
[19] 卢圣栋，现代分子生物学实验技术，高等教育出版社1993．
[20] 马凌波，张大兵等．甜菊糖基转移酶基因的克隆与分析．厦门大学学报(自然科学版)，2002，41(5)：531—535．
[21] 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．
[22] 欧阳光察，薛应龙．植物苯丙烷类代谢的生理意义及其调控．植物生理学通讯，1988(3)：9—16．
[23] 欧阳光察，应初衍等．植物苯丙氨酸解氨酶的研究Ⅵ．水稻、小麦PAL的纯化及基本特性．
植物生理学报，1985，11(2)：204—214．
[24] 秦佳梅．张弓．张卫东等．红景天叶片诱导再生植株．中国野生植物资源，1999，18(1)：45．
[25] 宋经元，张荫麟等，农杆菌介导的药用植物的转化．中国中药杂志，2000，25(3)：73—76．
[26] 孙敬三，桂耀林．植物细胞工程实验技术，科学出版社1995．
[27] 王关林，吴宏筠，植物基因工程原理与技术，科学出版社1998．
[28] 王敬文，薛应龙．植物苯丙氨酸解氨酶的研究—Ⅰ．植物激素对甘薯块根苯丙氨酸解氨酶和肉桂酸4—羟化酶活性变化及其伴随性的影响．植物生理学报，1981，7(4)：373—379．
[29] 王克夷．糖基转移酶的研究进展．生物化学与生物物理学进展，1994，21(1)：9—13．
[30] 王伟，钟英长．紫杉醇生物合成的研究．植物学通报，1999，16(2)：138—149．
[31] 王洋，张璞，于涛等．高效液相色谱法测定红景天甙含量方法的研究．植物研究，2001，21(1)：113—115．
[32] 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(博士论文)，东北林业大学，2001．
[33] 奚元龄，颜昌敬．植物细胞培养手册．农业出版社，1989，299—322．
[34] 邢建民，赵德修等．植物细胞培养生产黄酮类化合物研究进展．生物工程进展，2001，21(1)：47—50．
[35] 熊荣先．西藏红景天资源的调查．华西药学杂志，1995，8(10)：187-188．
[36] 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(博士论文)，大连理工大学，1996．
[37] 许建峰．韩爱明．冯朴荪等．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．
[38] 许建峰．韩爱明．冯朴荪等．高山红景天愈伤颗粒组织悬浮培养动力学及工艺的研究．生物工程学报，1996，12(4)：460—465．
[39] 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．
[40] 许建峰．应佩青．苏志国等．高山红景天资源应用于开发研究进展．中草药，1998，29(3)：202—205．
[41] 许建峰．赵岩．韩爱明等．高山红景天愈伤组织的诱导及培养研究．应用与环境生物学报,1995,1(1)19—25．
[42] 尹作鸿，朱蔚华．植物组织与细胞培养生产有用次生产物研究进展(Ⅰ)．中药材，1992，15(4)：43—45．
[43] 尹作鸿，朱蔚华．植物组织与细胞培养生产有用次生产物研究进展(Ⅱ)．中药材，1992，15(6)：42—44．
[44] 尹作鸿，朱蔚华．植物组织与细胞培养生产有用次生产物研究进展(Ⅲ)．中药材，1992，15
(7)：43—45．
[45] 印莉萍，刘祥林，分子细胞生物学实验技术，首都师范大学出版社2001．
[46] 于树宏，李玲等．利用细胞培养技术提高次生代谢物的产量．生物学通报，1998，33(7)40—42．
[47] 曾朝．红景天属植物药理作用的研究进展．湖北民族学院学报(医学版)，1999，16(4)：12—14．
[48] 张德炎，李喜文等．正交设计法在白三叶组织培养中的应用．东北师大学报(自然科学版)，1998，1：40—45．
[49] 赵剑，朱蔚华等．长春花生物碱合成途径和相关酶及其基因调控电研究进展．植物生理学通讯，1999，35(1)：60—68．
[50] 赵剑，朱蔚华等．长春花叶片愈伤组织诱导及培养过程中生物碱合成类型的变化和调节及有关酶的分析．中草药，1999，30(7)：533—537．
[51] 赵明强，丁家宜．植物培养物对外源化合物的糖基化．药学实践杂志，2000，18(5)：340．
[52] Annamraju D. Sarma, Yellamraju Sreelakshmi and Rameshwar Sharma. Diffrential Expression and Properties of Phenylalanine ammonia-lyase Isoforms in Tomato Leaves. Phytochemistry (1998) 49:2233-2243.
[53] Edwards R, Kessmann H (1992) ,Isoflavonoid phytoalexins and their biosynthetic enzymes.Molecular Plant Path—A Practical Approch IRL Prec. Oxford. UK.
[54] Ford C M,Boss P K, Bordier P .Cloning and characterization of Vitis vinifera UDP-Glucose: flavonoid 3-O- glucosyltransferase,a Homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in Vivo[J].The Journal of Biological Chemistry, 1998,273:9224-9233.
[55] Ganzera M,Yayla Y, Khan IA. Analysis of the marker compounds of Rhodiola rosea L. (golden root) by reversed phase high performance liquid chromatography. Chem Pharm Bull (Tokyo). 2001 Apr;49(4):465-7.
[56] H.A. Collin, G S. Eowaros. Plant cell culture.世界图书出版公司，1998．
[57] Han X,Zhang T, Wei Y, Cao X,Ito Y. Separation of salidroside from Rhodiola crenulata by high-speed counter-current chromatography. J Chromatogr A. 2002 Sep 20;971 (1-2):237-41.
[58] Hans-Jurgen JOOS and Klaus Hahl Brock. Phenylalanine ammonia-lyase in potato—Genomic complexity, structural comparison of two selected genes and modes of expression. Eur. J. Biochem. (1992) 2004:21-629.
[59] He-Ping Li, Rainer Fischer, Yu-Cai Liao. Molecular evidence for induction of phenylalanine ammonia-lyase during Puccinia graminis infection and elicitation in wheat. Molecular and physiological pathology,2001,23:286-291.
[60] Jones P R,Moller B L,Bordier Hoj P. The UDP-glucose: p-Hydroxymandeonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghun bicolor[J]. The Journal of Biological Chemistry, 1999,274:35483-35491.
[61] Kita M,Hirata Y, Moriguchi T, etal.Molecular cloning and characterization of a noval gene encoding iimonoid UDP- glucosyltransferrase in Citrus[J].FEBS Letters, 2000,469:173-178.
[62] Lee H,Raskin I,Purification,cloning, and expression of apathogen inducible UDP-glucose salicylic acid glucosyltransferrase from tobacco[J].The Journal of Biological Chemistry, 1999,274:36637-36642.
[63] Lei Y, Nan P, Tsering T, Bai Z,Tian C,Zhong Y. Chemical composition of the essential oils of two Rhodiola species from Tibet. Z Naturforsch [C]. 2003 Mar-Apr;58(3-4): 161-4.
[64] Li HB,Chen F. Preparative isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis by high-speed counter-current chromatography. J Chromatogr A. 2001 Oct 12;932(1-2):91-5.
[65] Luca Pellegrini,Odette Rohfritsch, Bernard Fritig and Michel Legrand. P henylalanine ammonia-lyase in Tobacco.Plant Physiol (1994) 106:877-886.
[66] Martin R,Mok M C,Mok D W S ,Isolation of a cytokinin gene,ZOG1,encoding zeatin Oglucosyltransferrase from Phaseolu lunatus[J].Proc.Natl.Acad. Sci. USA., 1999,96:284-289.
[67] Miroslawa Fuurmanowa, Malgorzata Hartwich, Augst W. Alfermann,Wiktor Kozminski&Mararian Olejnik, Rosavin as a product of glycosylation by Rhodiola rosea (roseroot) cell. Plant Cell,Tissue and Organ Culture, 1999,56:105-110.
[68] Shibata H,Sawa Y, Oka T, etal. Steviol and steviolglycoside:glucosyltransferase activities in Stevia rebaudiana Bertoni-purificatio and partial characterizeation [J].Archives of Biochemistry and Biophysics, 1995,321:390-396.
[69] Wenzhe Fan,Yasuhiro Tezuka Khin May Ni,and Shigetoshi Kadota, Prolyl Endopeptidase Inhibitors from the Underground Part of Rhodiola sachalinensis .Chem .Pharm. Bull.2001,49(4):396-401.
[70] Yamazaki M,Gong Z,Fukuchi Mizutani M,etal.Molecular cloning and biochemical characterizrtion of a novel anthocyanin 5-O-glucosyltransferrase by mRNA differential display for plant forms regarding anthocyanin[J].The Journal of Biological Chemistry, 1999,274:7405-7411.
[71] Yoshikawa M,Shimada H,Shimoda H,Murakami N,Yamahara J,Matsuda H. ioactive constituents of Chinese natural medicines. Ⅱ. Rhodiolae radix. (1). Chemical structures and antiallergic activity of rhodiocyanosides A and B from the underground part of Rhodiola quadrifida (Pall.)Fisch. et Mey. (Crassulaceae).Chem Pharm Bull (Tokyo). 1996 Nov;44(11):2086-91.
[2] 陈敏，廖志华等．生物技术在长春花研究中的应用．中草药，2001，32(1)：78—81．
[3] 陈书安，王晓东等．藏红花的研究进展．中草药，2001，32(12)：1137—1139．
[4] 陈晓亚，刘培等．植物次生代谢的分子生物学及基因工程．生命科学，1996，8(2)：8—10．
[5] 陈晓亚，刘培等．植物的次生代谢．科学，1996，48(5)：37—39．
[6] 成静，郭勇．植物细胞工程药物生产的研究进展．江西科学，2000，18(1)：60—62．
[7] 崔堂兵，郭勇等．植物组织培养中褐变现象的产生机理及克服方法．广东农业科学，2001，3：16—18．
[8] 戴良先，川西高原红景天产业化开发的思考．四川草原，2000，2：61—64．
[9] 高国训．植物组织培养中的褐变问题．植物生理学通讯，1999，35(6)：501—506．
[10] 韩爱明．许建峰．方晓丹等．影响高山红景天细胞悬浮培养中细胞生长和红景天甙积累的几个因素．植物生理学通讯1997,33(1)：33-36．
[11] 胡国武，温廷益等．紫杉醇合成代谢途径中紫杉烯合成酶cDNA的克隆．生物工程学报，2000，16(2)：157—159．
[12] 胡维，刘敬忠等．苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达及酶法合成L-苯丙氨酸．微生物学杂志，2000，20(3)：30—32．
[13] 贾冬梅，李春荣等．高山红景天开发概述与应用开发建议．黑河科技，2000，4：6—7．
[14] 贾国夫．红景天的研究概况及展望．四川草原，1997，3：38—40．
[15] 江昌俊，余有本．苯丙氨酸解氨酶的研究进展．安徽农业大学学报，2001，28(4)：425—430．
[16] 姜明兰．钟文田．韩洪等．组织培养生产高山红景天有效成分的研究Ⅰ．高山红景天愈伤组织的诱导与培养．沈阳农业大学学报，1994,25(4)：355—359．
[17] 李永明，赵玉琪等．实用分子生物学方法手册，科学出版社．1998．
[18] 刘春朝，王玉春等．生物诱导子调节植物组织次生代谢的研究．植物学通报，1999，16(2)131—137．
[19] 卢圣栋，现代分子生物学实验技术，高等教育出版社1993．
[20] 马凌波，张大兵等．甜菊糖基转移酶基因的克隆与分析．厦门大学学报(自然科学版)，2002，41(5)：531—535．
[21] 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．
[22] 欧阳光察，薛应龙．植物苯丙烷类代谢的生理意义及其调控．植物生理学通讯，1988(3)：9—16．
[23] 欧阳光察，应初衍等．植物苯丙氨酸解氨酶的研究Ⅵ．水稻、小麦PAL的纯化及基本特性．
植物生理学报，1985，11(2)：204—214．
[24] 秦佳梅．张弓．张卫东等．红景天叶片诱导再生植株．中国野生植物资源，1999，18(1)：45．
[25] 宋经元，张荫麟等，农杆菌介导的药用植物的转化．中国中药杂志，2000，25(3)：73—76．
[26] 孙敬三，桂耀林．植物细胞工程实验技术，科学出版社1995．
[27] 王关林，吴宏筠，植物基因工程原理与技术，科学出版社1998．
[28] 王敬文，薛应龙．植物苯丙氨酸解氨酶的研究—Ⅰ．植物激素对甘薯块根苯丙氨酸解氨酶和肉桂酸4—羟化酶活性变化及其伴随性的影响．植物生理学报，1981，7(4)：373—379．
[29] 王克夷．糖基转移酶的研究进展．生物化学与生物物理学进展，1994，21(1)：9—13．
[30] 王伟，钟英长．紫杉醇生物合成的研究．植物学通报，1999，16(2)：138—149．
[31] 王洋，张璞，于涛等．高效液相色谱法测定红景天甙含量方法的研究．植物研究，2001，21(1)：113—115．
[32] 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(博士论文)，东北林业大学，2001．
[33] 奚元龄，颜昌敬．植物细胞培养手册．农业出版社，1989，299—322．
[34] 邢建民，赵德修等．植物细胞培养生产黄酮类化合物研究进展．生物工程进展，2001，21(1)：47—50．
[35] 熊荣先．西藏红景天资源的调查．华西药学杂志，1995，8(10)：187-188．
[36] 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(博士论文)，大连理工大学，1996．
[37] 许建峰．韩爱明．冯朴荪等．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．
[38] 许建峰．韩爱明．冯朴荪等．高山红景天愈伤颗粒组织悬浮培养动力学及工艺的研究．生物工程学报，1996，12(4)：460—465．
[39] 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．
[40] 许建峰．应佩青．苏志国等．高山红景天资源应用于开发研究进展．中草药，1998，29(3)：202—205．
[41] 许建峰．赵岩．韩爱明等．高山红景天愈伤组织的诱导及培养研究．应用与环境生物学报,1995,1(1)19—25．
[42] 尹作鸿，朱蔚华．植物组织与细胞培养生产有用次生产物研究进展(Ⅰ)．中药材，1992，15(4)：43—45．
[43] 尹作鸿，朱蔚华．植物组织与细胞培养生产有用次生产物研究进展(Ⅱ)．中药材，1992，15(6)：42—44．
[44] 尹作鸿，朱蔚华．植物组织与细胞培养生产有用次生产物研究进展(Ⅲ)．中药材，1992，15
(7)：43—45．
[45] 印莉萍，刘祥林，分子细胞生物学实验技术，首都师范大学出版社2001．
[46] 于树宏，李玲等．利用细胞培养技术提高次生代谢物的产量．生物学通报，1998，33(7)40—42．
[47] 曾朝．红景天属植物药理作用的研究进展．湖北民族学院学报(医学版)，1999，16(4)：12—14．
[48] 张德炎，李喜文等．正交设计法在白三叶组织培养中的应用．东北师大学报(自然科学版)，1998，1：40—45．
[49] 赵剑，朱蔚华等．长春花生物碱合成途径和相关酶及其基因调控电研究进展．植物生理学通讯，1999，35(1)：60—68．
[50] 赵剑，朱蔚华等．长春花叶片愈伤组织诱导及培养过程中生物碱合成类型的变化和调节及有关酶的分析．中草药，1999，30(7)：533—537．
[51] 赵明强，丁家宜．植物培养物对外源化合物的糖基化．药学实践杂志，2000，18(5)：340．
[52] Annamraju D. Sarma, Yellamraju Sreelakshmi and Rameshwar Sharma. Diffrential Expression and Properties of Phenylalanine ammonia-lyase Isoforms in Tomato Leaves. Phytochemistry (1998) 49:2233-2243.
[53] Edwards R, Kessmann H (1992) ,Isoflavonoid phytoalexins and their biosynthetic enzymes.Molecular Plant Path—A Practical Approch IRL Prec. Oxford. UK.
[54] Ford C M,Boss P K, Bordier P .Cloning and characterization of Vitis vinifera UDP-Glucose: flavonoid 3-O- glucosyltransferase,a Homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in Vivo[J].The Journal of Biological Chemistry, 1998,273:9224-9233.
[55] Ganzera M,Yayla Y, Khan IA. Analysis of the marker compounds of Rhodiola rosea L. (golden root) by reversed phase high performance liquid chromatography. Chem Pharm Bull (Tokyo). 2001 Apr;49(4):465-7.
[56] H.A. Collin, G S. Eowaros. Plant cell culture.世界图书出版公司，1998．
[57] Han X,Zhang T, Wei Y, Cao X,Ito Y. Separation of salidroside from Rhodiola crenulata by high-speed counter-current chromatography. J Chromatogr A. 2002 Sep 20;971 (1-2):237-41.
[58] Hans-Jurgen JOOS and Klaus Hahl Brock. Phenylalanine ammonia-lyase in potato—Genomic complexity, structural comparison of two selected genes and modes of expression. Eur. J. Biochem. (1992) 2004:21-629.
[59] He-Ping Li, Rainer Fischer, Yu-Cai Liao. Molecular evidence for induction of phenylalanine ammonia-lyase during Puccinia graminis infection and elicitation in wheat. Molecular and physiological pathology,2001,23:286-291.
[60] Jones P R,Moller B L,Bordier Hoj P. The UDP-glucose: p-Hydroxymandeonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghun bicolor[J]. The Journal of Biological Chemistry, 1999,274:35483-35491.
[61] Kita M,Hirata Y, Moriguchi T, etal.Molecular cloning and characterization of a noval gene encoding iimonoid UDP- glucosyltransferrase in Citrus[J].FEBS Letters, 2000,469:173-178.
[62] Lee H,Raskin I,Purification,cloning, and expression of apathogen inducible UDP-glucose salicylic acid glucosyltransferrase from tobacco[J].The Journal of Biological Chemistry, 1999,274:36637-36642.
[63] Lei Y, Nan P, Tsering T, Bai Z,Tian C,Zhong Y. Chemical composition of the essential oils of two Rhodiola species from Tibet. Z Naturforsch [C]. 2003 Mar-Apr;58(3-4): 161-4.
[64] Li HB,Chen F. Preparative isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis by high-speed counter-current chromatography. J Chromatogr A. 2001 Oct 12;932(1-2):91-5.
[65] Luca Pellegrini,Odette Rohfritsch, Bernard Fritig and Michel Legrand. P henylalanine ammonia-lyase in Tobacco.Plant Physiol (1994) 106:877-886.
[66] Martin R,Mok M C,Mok D W S ,Isolation of a cytokinin gene,ZOG1,encoding zeatin Oglucosyltransferrase from Phaseolu lunatus[J].Proc.Natl.Acad. Sci. USA., 1999,96:284-289.
[67] Miroslawa Fuurmanowa, Malgorzata Hartwich, Augst W. Alfermann,Wiktor Kozminski&Mararian Olejnik, Rosavin as a product of glycosylation by Rhodiola rosea (roseroot) cell. Plant Cell,Tissue and Organ Culture, 1999,56:105-110.
[68] Shibata H,Sawa Y, Oka T, etal. Steviol and steviolglycoside:glucosyltransferase activities in Stevia rebaudiana Bertoni-purificatio and partial characterizeation [J].Archives of Biochemistry and Biophysics, 1995,321:390-396.
[69] Wenzhe Fan,Yasuhiro Tezuka Khin May Ni,and Shigetoshi Kadota, Prolyl Endopeptidase Inhibitors from the Underground Part of Rhodiola sachalinensis .Chem .Pharm. Bull.2001,49(4):396-401.
[70] Yamazaki M,Gong Z,Fukuchi Mizutani M,etal.Molecular cloning and biochemical characterizrtion of a novel anthocyanin 5-O-glucosyltransferrase by mRNA differential display for plant forms regarding anthocyanin[J].The Journal of Biological Chemistry, 1999,274:7405-7411.
[71] Yoshikawa M,Shimada H,Shimoda H,Murakami N,Yamahara J,Matsuda H. ioactive constituents of Chinese natural medicines. Ⅱ. Rhodiolae radix. (1). Chemical structures and antiallergic activity of rhodiocyanosides A and B from the underground part of Rhodiola quadrifida (Pall.)Fisch. et Mey. (Crassulaceae).Chem Pharm Bull (Tokyo). 1996 Nov;44(11):2086-91.