干涉丹参SmORA1对植物抗病和丹参酮类次生代谢的影响

摘要

Objective]ERF (ethylene responsive factor) are a class of plant-specific transcription factors, which are generally involved in plant stress responses. An ERF gene (namedSmORA1) has been screened from medicinal plantSalvia miltiorrhiza, which is highly homologous toCrORCA3 ofCatharanthus roseus. The objective of this study is to investigate the effects ofSmORA1 on the resistance to disease and on the regulation of tanshinone biosynthesis inS. miltiorrhiza through interference ofSmORA1 expression inS. miltiorrhiza by RNAi method.[Method]SmORA1was cloned and inserted into interference vector, then the constructed vector (pk-ORAi) was transformed into S. miltiorrhiza byAgrobacterium tumefaciens-mediated method and transgenic lines ofSmORA1-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 SmORA1-RNAi transgenic lines were detected after infection byRhizoctonia solani. The concentrations of tanshinones including dihydrotanshinone, cryptotanshinone and tanshinone II A inSmORA1-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 ofSmORA1 were obtained after antibiotics and real-time qPCR screening, interference efficiency achieved at 80%. Afterinfection withR. solani, the disease indexes ofSmORA1-RNAi lines were significantly higher than those of wild type (WT) and empty vector (VK). The contents of MDA inSmORA1-RNAi lines were also significantly higher than those of control lines, which indicated that the anti-aging ability of transgenic lines was significantly decreased. The activities of SOD, CAT and PAL inSmORA1-RNAi lines were significantly lower than those of the control. The contents of Pro and GSH were also dramatically decreased inSmORA1-RNAi lines. Moreover, the expression levels ofSmPDF1.2, SmSTH-2andSmPR-10 inSmORA1-RNAi lines were down-regulated compared with the control lines. These results implied that SmORA1 play important roles in plant resistance. Furthermore, the expression ofSmHMGR1,SmHMGR2,SmGPPS,SmGGPPS1 andSmGGPPS3,encoding key enzymes involved tanshinone biosynthesis, were down-regulated inSmORA1-RNAi lines. Meanwhile, the contents of tanshinoneⅡA and cryptotanshinone were also reduced inSmORA1-RNAi lines compared with the control lines. So it was speculated that SmORA1regulated tanshinone biosynthesis throught the regulation of the expression ofSmHMGR1, SmHMGR2,SmGPPS,SmGGPPS1 andSmGGPPS3.[Conclusion]SmORA1 is involved in the resistance response and closely related to the regulation of tanshinone biosynthesis inS. miltiorrhiza.%【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CrORCA3高度同源的ERF转录因子编码基因（命名为SmORA1）。论文旨在通过干涉丹参SmORA1的表达，进一步明确SmORA1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SmORA1基因片段，构建SmORA1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SmORA1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SmORA1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SmORA1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，SmORA1-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；SmORA1-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SmORA1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SmORA1的丹参株系抗病性也呈现明显减弱；进一步研究发现SmORA1-RNAi转基因株系中抗病性相关蛋白编码基因SmPDF1.2、SmSTH-2和SmPR-10的表达量明显低于对照株系。采用HPLC方法分析发现，丹参SmORA1干涉株系中丹参酮ⅡA和隐丹参酮含量显著下降；同时采用实时定量PCR检测分析，发现丹参SmORA1干涉株系中丹参酮合成途径关键酶基因SmHMGR1、SmHMGR2、SmGPPS、SmGGPPS1和SmGGPPS3等表达显著下调,说明SmORA1可能通过调节SmHMGR1、SmHMGR2、SmGPPS、SmGGPPS1和SmGGPPS3等丹参酮合成途径关键酶基因的表达参与丹参酮类化合物的合成调控。【结论】丹参SmORA1参与了丹参抗病反应，而且与丹参酮类次生代谢物质的合成密切相关。