干涉丹参SmORA1对植物抗病和丹参酮类次生代谢的影响  

Effect of RNAi of Sm ORA1 on Disease Resistance and Tanshinones Secondary Metabolism in Salvia miltiorrhiza

作　　者：化文平[1,2] 刘文超[1] 王喆之[1] 李翠芹[1] 

机构地区：[1]陕西师范大学药用资源与天然药物化学教育部重点实验室/西北濒危药材资源开发国家工程实验室/生命科学学院,西安710062 [2]陕西学前师范学院生物科学与技术系,西安710061

出　　处：《中国农业科学》2016年第3期491-502,共12页Scientia Agricultura Sinica

基　　金："十二五"国家科技支撑计划(2011BA106B06);中央高校基本科研业务费专项资金(GK201303009);陕西省自然科学基金(2014JQ3105);陕西学前师范学院基金(2014DS010)

摘　　要：【目的】乙烯响应因子(ethylene response factor,ERF)是植物特有的一类转录因子,普遍参与植物各类胁迫反应。前期从药用植物丹参(Salvia miltiorrhiza)中筛选得到了一条与长春花(Catharanthus roseus)中Cr ORCA3高度同源的ERF转录因子编码基因(命名为Sm ORA1)。论文旨在通过干涉丹参Sm ORA1的表达,进一步明确Sm ORA1在植物抗病反应与丹参酮合成代谢调控中的作用。【方法】扩增Sm ORA1基因片段,构建Sm ORA1基因干涉载体pk-ORAi并采用农杆菌介导的叶盘转化法转化丹参,经抗生素抗性筛选和PCR鉴定获得阳性转基因丹参植株;采用分光光度法或酶联免疫方法检测立枯丝核菌(Rhizoctonia solani)侵染后转基因株系中丙氨酸解氨酶(phenylalnine ammonialyase,PAL)、过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)等抗性相关酶活性和还原型谷胱甘肽(glutathione,GSH)、脯氨酸(proline,Pro)、丙二醛(malondialdehyde,MDA)含量等抗性相关生理指标的变化;采用HPLC法考察干涉Sm ORA1表达对二氢丹参酮、隐丹参酮和丹参酮ⅡA等丹参酮类次生代谢物含量的影响;采用实时定量PCR考察干涉Sm ORA1对丹参株系抗性基因和丹参酮合成相关关键酶基因表达的影响。【结果】经抗生素和PCR筛选获得了11个阳性株系,进一步采用实时定量PCR分析得到3个Sm ORA1表达显著下调的转基因株系(RNAi-11、RNAi-18、RNAi-22),干涉效率达到80%以上。立枯丝核菌对丹参植株有明显的致病力,可以有效诱导丹参发病;其侵染后,Sm ORA1-RNAi转基因株系病情指数显著高于野生型(WT)和空载(VK)对照株系,病情更严重;Sm ORA1-RNAi转基因株系中的MDA含量显著高于WT和VK对照株系,说明干涉Sm ORA1的丹参株系抗衰老能力显著下降;植物抗性相关的PAL、CAT和SOD等酶活性以及GSH和Pro等物质含量显著低于对照株系,说明干涉Sm ORA1的丹参株系抗病性也呈现明显减弱;进一步研究[ Objective ] ERF （ethylene responsive factor） are a class of plant-specific transcription factors, which are generally involved in plant stress responses. An ERF gene （named SmORA1） has been screened from medicinal plant Salvia miltiorrhiza, which is highly homologous to CrORCA3 of Catharanthus roseus. The objective of this study is to investigate the effects of SrnORA1 on the resistance to disease and on the regulation of tanshinone biosynthesis in S. miltiorrhiza through interference of SmORA1 expression in S. miltiorrhiza by RNAi method. [Method] SmORA1 was cloned and inserted into interference vector, then the constructed vector （pk-ORAi） was transformed into S. miltiorrhiza by Agrobacterium tumefaciens-mediated method and transgenic lines of SmORA1-RNAi were obtained by antibiotic resistance screening and PCR identification. Using the spectrophotometry or enzyme linked immunosorbent assay, the resistance-related enzymes （phenylalnine ammonialyase （PAL）, catalase （CAT） and superoxide dismutase （SOD）） activities and physiological indexes （glutathione （GSH）, proline （Pro） and malondialdehyde （MDA）） in SrnORA1-RNAi transgenic lines were detected after infection by Rhizoctonia solani. The concentrations of tanshinones including dihydrotanshinone, cryptotanshinone and tanshinone ⅡA in SmORA1-RNAi transgenic lines were detected using HPLC method. The expression levels of genes related to resistance and involved in tanshinone biosynthesis were investigated by real-time qPCR. [ Result ] A total of 11 positive transgenic lines were got after antibiotics and PCR screening, among of which, three lines （RNAi- 11, RNAi-18, RNAi-22） with significantly down-regulated expression levels of SmORA1 were obtained after antibiotics and real-time qPCR screening, interference efficiency achieved at 80%. After infection with R. solani, the disease indexes of SmORA1-RNAi lines were significantly higher than those of wild type （WT） and empty vector （VK）. The contents of MDA i

关 键 词：丹参 乙烯响应因子 抗病性 丹参酮 

分 类 号：S567.53[农业科学—中草药栽培]