银杏萜内酯代谢途径中关键酶基因的遗传转化研究
摘要
银杏萜内酯,包括银杏内酯和白果内酯,是在银杏提取物中起着主要作用的有效药用成分,其独特的生理作用和治疗价值在世界医疗行业引起了极大兴趣。然而,银杏萜内酯在银杏中含量较低,且银杏的细胞培养系很少产银杏萜内酯。由于不断扩大的银杏制品的市场需求和有限的银杏树资源,用基因工程技术修饰银杏萜内酯生物合成途径已成为提高银杏萜内酯含量最具潜力的方法之一。
法尼基焦磷酸合成酶(FPS)是异戊烯基转移酶中的一种,它催化一分子的二甲基烯丙基焦磷酸(DMAPP)和两分子的异戊烯基焦磷酸(IPP)缩合生成法尼基焦磷酸(FPP)。FPP是植物体内甾体、皂苷、倍半萜、橡胶等许多萜类衍生物质的合成前体。在银杏中,FPP为合成白果内酯合成提供前体,同时也为香叶基香叶基焦磷酸(GGPP)的生成提供直接前体,而GGPP是包括银杏内酯在内的所有二萜类化合物的共同前体。1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶(HDR)是MEP途径末端的酶,以5:1的比例同时把1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸(HMBPP)转化成IPP和DMAPP的混合物,为银杏萜内酯的生物合成提供了异戊烯前体。FPS和HDR都是银杏内酯合成途径中的关键酶。因此,fps基因和hdr基因也成为了研究银杏内酯代谢工程的两个潜在靶点。
为了研究FPS和HDR对银杏愈伤中银杏内酯含量的影响,我们基于pCAMBIA1304载体构建了两个单基因表达载体,分别是载体p1304+Gbfps和载体p1304+Gbhdr。采用根癌农杆菌介导法,分别将Gbfps和Gbhdr转化银杏种子胚。通过潮霉素(10mg/L)筛选,分别获得了32个和8个抗性愈伤组织系,PCR检测表明其中分别有30个和7个为转基因愈伤组织系。采用HPLC-ELSD法分别对转Gbfps的22个愈伤组织和转Gbhdr的4个愈伤组织中的银杏总内酯含量进行了测定。结果显示,转Gbfps的愈伤组织系f-6中银杏总内酯含量相比非转基因系有大幅提高,达到非转基因系的4.4倍,转Gbhdr的愈伤组织系h-8中银杏总内酯含量达到非转基因系的2.5倍。
Ginkgolides are pharmaceutical effective ingredients playing major part in the established medicinal functions of Ginkgo extracts,which have attracted great commercial interests as pharmaceuticals or nutraceuticals.However the contents of ginkgolides are very low in the native ginkgo plant materials and the ginkgo cell cultures hardly produce ginkgolides.Because of the expanding demands for ginkgo products and limited ginkgo tree resources,genetic modification of the ginkgolides biosynthesis pathway is becoming one of the most potential ways to increase ginkgolides content.
Farnesyl diphosphate synthase(FPS) catalyzes two consecutive condensations of isopentenyl diphosphate with dimethylallyl diphosphate,and the resultant ger-anyl diphosphate.The ultimate product of these two reactions,arnesyl diphosphate(FPP), provides precursor for the biosynthesis of sterols,dolichols,sesquiterpene trilactone, prenylated proteins in plant.In Ginkgo biloba,it provides precursor for Bilobalide biosynthesis.1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR) is proved to be the terminal-acting enzyme in the plastid MEP pathway which provides isoprenoid precursors for the biosynthesis of ginkgolides.FPS and HDR are the two key-enzymes in the ginkgolides biosynthesis pathway.So we choose the Gbfps and Gbhdr as the target genes for further study in metabolic engineering of ginkgolides.
In order to investigate the effects of the two enzymes on the yields of ginkgolides in ginkgo calli,we construct two vectors of gene expression based on pCAMBIA1304 for transformation:Vector p1304+ Gbfps and Vector p1304+Gbhdr.These two genes were successfully transferred into embryos of Ginkgo biloba with Agrobacterium tumefaciens-mediated transformation method.After hygromycin(10 mg/L) selection, 32 and 8 hygromycin-resistant calli were obtained respectly.PCR analysis confirmed the Gbfps transgenic status of thirty independent callus lines and Gbhdr transgenic status of seven independent callus lines.Ginkgolides contents in 22 independent Gbfps transgenic callus lines and 4 independent Gbhdr transgenic callus lines were detected by HPLC-ELSD analysis respectly,and the result showed that ginkgolides content in line f-6 was significantly increased,reaching the 4.4 fold of the untransformed control line;ginkgolides content in line h-8 reached the 2.5 fold of the untransformed control line.
法尼基焦磷酸合成酶(FPS)是异戊烯基转移酶中的一种,它催化一分子的二甲基烯丙基焦磷酸(DMAPP)和两分子的异戊烯基焦磷酸(IPP)缩合生成法尼基焦磷酸(FPP)。FPP是植物体内甾体、皂苷、倍半萜、橡胶等许多萜类衍生物质的合成前体。在银杏中,FPP为合成白果内酯合成提供前体,同时也为香叶基香叶基焦磷酸(GGPP)的生成提供直接前体,而GGPP是包括银杏内酯在内的所有二萜类化合物的共同前体。1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶(HDR)是MEP途径末端的酶,以5:1的比例同时把1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸(HMBPP)转化成IPP和DMAPP的混合物,为银杏萜内酯的生物合成提供了异戊烯前体。FPS和HDR都是银杏内酯合成途径中的关键酶。因此,fps基因和hdr基因也成为了研究银杏内酯代谢工程的两个潜在靶点。
为了研究FPS和HDR对银杏愈伤中银杏内酯含量的影响,我们基于pCAMBIA1304载体构建了两个单基因表达载体,分别是载体p1304+Gbfps和载体p1304+Gbhdr。采用根癌农杆菌介导法,分别将Gbfps和Gbhdr转化银杏种子胚。通过潮霉素(10mg/L)筛选,分别获得了32个和8个抗性愈伤组织系,PCR检测表明其中分别有30个和7个为转基因愈伤组织系。采用HPLC-ELSD法分别对转Gbfps的22个愈伤组织和转Gbhdr的4个愈伤组织中的银杏总内酯含量进行了测定。结果显示,转Gbfps的愈伤组织系f-6中银杏总内酯含量相比非转基因系有大幅提高,达到非转基因系的4.4倍,转Gbhdr的愈伤组织系h-8中银杏总内酯含量达到非转基因系的2.5倍。
Ginkgolides are pharmaceutical effective ingredients playing major part in the established medicinal functions of Ginkgo extracts,which have attracted great commercial interests as pharmaceuticals or nutraceuticals.However the contents of ginkgolides are very low in the native ginkgo plant materials and the ginkgo cell cultures hardly produce ginkgolides.Because of the expanding demands for ginkgo products and limited ginkgo tree resources,genetic modification of the ginkgolides biosynthesis pathway is becoming one of the most potential ways to increase ginkgolides content.
Farnesyl diphosphate synthase(FPS) catalyzes two consecutive condensations of isopentenyl diphosphate with dimethylallyl diphosphate,and the resultant ger-anyl diphosphate.The ultimate product of these two reactions,arnesyl diphosphate(FPP), provides precursor for the biosynthesis of sterols,dolichols,sesquiterpene trilactone, prenylated proteins in plant.In Ginkgo biloba,it provides precursor for Bilobalide biosynthesis.1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR) is proved to be the terminal-acting enzyme in the plastid MEP pathway which provides isoprenoid precursors for the biosynthesis of ginkgolides.FPS and HDR are the two key-enzymes in the ginkgolides biosynthesis pathway.So we choose the Gbfps and Gbhdr as the target genes for further study in metabolic engineering of ginkgolides.
In order to investigate the effects of the two enzymes on the yields of ginkgolides in ginkgo calli,we construct two vectors of gene expression based on pCAMBIA1304 for transformation:Vector p1304+ Gbfps and Vector p1304+Gbhdr.These two genes were successfully transferred into embryos of Ginkgo biloba with Agrobacterium tumefaciens-mediated transformation method.After hygromycin(10 mg/L) selection, 32 and 8 hygromycin-resistant calli were obtained respectly.PCR analysis confirmed the Gbfps transgenic status of thirty independent callus lines and Gbhdr transgenic status of seven independent callus lines.Ginkgolides contents in 22 independent Gbfps transgenic callus lines and 4 independent Gbhdr transgenic callus lines were detected by HPLC-ELSD analysis respectly,and the result showed that ginkgolides content in line f-6 was significantly increased,reaching the 4.4 fold of the untransformed control line;ginkgolides content in line h-8 reached the 2.5 fold of the untransformed control line.
引文
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李树钧,孙滨.银杏苦内酯B对哮喘豚鼠气道反应性和嗜酸性粒细胞的影响[J].第四军医大学学报,1995,16:269.
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杜文华,陈惠孙,胡德耀,等.银杏苦内酯B对家兔内毒素休克心功能抑制的防治作用研究[J].中药药理与临床,1996,12:11.
杜文华,陈惠孙,陆松敏,胡德耀,李叶君,牟江洪,周萍.PAF受体拮抗剂苦银杏内酯B对内毒素休克不同介质的作用[J].中药药理与临床,1997,13:21-23.
李明,高向东.吴梧桐.银杏内酯对NaCN及缺糖引起的PC12细胞凋亡的保护作用[J].药物生物技术,2000,7:46-49.
李树钧,孙滨.银杏苦内酯B对哮喘豚鼠气道反应性和嗜酸性粒细胞的影响[J].第四军医大学学报,1995,16:269.
李新岗,陈薇,韦玮,等.银杏叶中银杏内酯的实验室提取方法研究.中国医药工业杂志,1998,29:7-8.叶敏,果德安.银杏萜内酯的研究概况[J].世界科学技术中医药现代化,2003,5:33-37.
刘稚,樊梦康,崔伟.土壤根癌杆菌体外转化胡萝卜悬浮培养细胞[J].中国科学(B 辑),1987:507-512.
肖建如,侯铁胜,陆永坚,等.银杏苦内酯治疗急性颈髓损伤患者疗效观察[J].中国危重病急救医学,1998,10:218.
周龙恩,王文杰,白金叶,等.银杏内酯B对血小板活化因子激活的大鼠中性白细胞功能的影响[J].中草药,2000,13:528-31.
朱业晋,张杰.HPLC-ELSD在药物分析中的应用进展[J].赣南师范学院学报,2003,6:39-41.
Adam P,Hecht S,Eisenreich W,Kaiser J,Grawert T,Arigoni D,Bacher A,Rohdich F.Biosynthesis of terpenes:studies on 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase[J].Proceedings of National Academy of Sciences of the United States of America,2002,99:12108-12113.
Amri H,Ogwuegbu SO.In vivo regulation of peripheral type benzodiazepine receptor ang glucocorticoid synthesis by Ginkgo biloba extract EGb 761 and isolated ginkgolieds[J].Endocrtnology,1996,137:5707-5718.
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