茉莉酸调节绞股蓝人参皂苷类物质积累的研究

Jiao-Gu-Lan (Gynostemma pentaphyllum (Thunb.) Makino) belongs to the genus of Gynostemma, Cucurbitaceae. Due to its capability of synthesizing and accumulating ginsenosides which are well known medicinal triterpenes benefiting human health, it has been honored as "Southern Ginseng", and therefore, is increasingly becoming an important officinal plant. Currently, this medicinal plant is widely grown in south China areas to produce different beneficial supplements. It is predicted that supplement products of this plant will likely form equivalents of ginseng. Based on the facts that light can affect the structure, development and accumulation of ginsenosides in G. pentaphyllum, additionally, jasmonate has been reported a key signal molecule in regulating the accumulation of the plant secondary metabolites, and exogenous jasmonic acid can enhance the accumulation of ginsenosides in cell cultures of ginseng genus plants, therefore, we propose that the effects of light on formation of ginsenosides in G. pentaphyllum are through a mechanism by which jasmonte controls the biosynthetic pathway. In this project, we propose to complete cell biology, phytochemistry and molecular biology studies to understand mechanisms by which jasmonate regulates ginsenoides in tissues of G. pentaphyllum. Expereiments will include effects of light intensity and light quality on production of jasmonic acid, gene expression and enzyme activities involved in squalene synthase and squalene epoxidase, and production of gypenosides in G. pentaphyllum. In addition, we will apply exogenous methyl jasmonate to plants to understand the effects on production of ginsenosides. Furthermore, we will perform experiments of immunological localization for both squalene synthase and squalene epoxidase to determine the location of gypenosieds biosynthesis in G. pentaphyllum. The results will enhance the understanding of regulation relevance of light, jasmonate and ginsenodies. This accomplishment will further enhance the development of agricultural technologies of G. pentaphyllum for the final purpose of field growth.

绞股蓝为葫芦科绞股蓝属植物，因体内能合成和积累人参皂苷类物质而被称为"南方人参"，是一种重要的药用植物。绞股蓝在我国南方适宜种植的地域广，生产潜力大，有望成为人参的代用品。基于光对绞股蓝的结构发育和人参皂苷类物质积累具有重要影响，而茉莉酸又是调节植物次生代谢产物积累的关键信号分子，且外源茉莉酸能够促进人参属植物细胞积累人参皂苷，我们推测光对绞股蓝中人参皂苷类物质的影响是通过茉莉酸途径来起调控作用的。本项目拟采用细胞生物学、植物化学和分子生物学的相关技术，研究光强、光质对绞股蓝内源茉莉酸含量、绞股蓝中人参皂苷生物合成关键酶鲨烯合成酶和鲨烯环氧酶的基因表达、酶活性和皂苷含量的影响，并用施加外源茉莉酸甲酯的生理效应来验证这一设想。实验通过对两种关键酶免疫定位的方法来确立绞股蓝人参皂苷的合成场所。本研究试图阐明茉莉酸对绞股蓝人参皂苷类物质积累的调节机制，为发展绞股蓝的高效栽培技术提供理论依。

项目完成了对绞股蓝人参皂苷生物合成关键酶鲨烯环氧酶基因GpSE1和鲨烯合成酶基因GpSS1的克隆及序列分析，对GpSE1和GpSS1进行了原核表达载体构建和蛋白诱导表达，并进行了进化分析。基于GpSE1多克隆抗体制备和抗体免疫特异性分析的免疫荧光定位，绞股蓝叶肉细胞中的GpSE1荧光信号为多边形网状结构，与已报道的拟南芥内质网结合蛋白的定位模式一致。对绞股蓝幼叶、老叶及根状茎中GpSE1 基因和GpSS1基因表达水平的检测显示，它们均在幼叶中表达水平最高，老叶中的表达水平次之，根状茎最低，表明幼叶是合成皂苷物质的主要场所。.研究发现，光对绞股蓝人参皂苷含量和两种关键酶基因表达具有显著影响。皂苷含量在红光下最高，蓝光次之，白光高于黑暗。而相对应光质下的GpSE1基因和GpSS1的基因表达水平呈类似的趋势。统计分析说明不同光质下的皂苷含量与两种基因表达水平呈显著的正相关关系，暗示光质是通过改变关键酶基因表达而调节皂苷含量的。同时，光质还影响绞股蓝的生长和光合作用。白光促进生物量、茎粗和新叶萌发，其净光合速率也最高，但红光下的皂苷含量最高，绿光的光合效率最低。说明光质对生长和皂苷积累的影响与光合作用效率直接相关。.光质对皂苷积累的调节可能是通过茉莉酸信号途径。不同浓度的茉莉酸甲酯（MeJA）喷施植株后均引起GpSE1基因和GpSS1 基因的表达上调，其中50 μmol/L的MeJA 对基因表达的影响最大。两个基因在幼叶和老叶中的表达均呈现出先逐渐上升后有所下降的趋势，且在幼叶中的表达水平高于老叶。GpSE1基因在MeJA处理后的72 h达到峰值，而GpSS1基因表达上调的峰值在MeJA处理后的48h。.此外，在基金支持下，还完成了绞股蓝种子油的提取、成分分析和急性毒性实验，短时低温对绞股蓝种子萌发的促进作用，不同居群五柱绞股蓝叶形变异及总黄酮含量分析，绞股蓝属三种植物的营养成分分析，具有ACC脱氨酶活性的绞股蓝根际细菌的分离鉴定及其促生作用等相关研究工作。