草地贪夜蛾取食诱导下玉米酵母双杂交文库的构建及ZmRop1互作蛋白筛选

Construction of library of maize yeast two-hybrid induced by Spodoptera frugiperda feeding and screening of ZmRop1-interacting proteins

【目的】构建以草地贪夜蛾取食诱导后的玉米酵母cDNA文库以及编码植物Rac蛋白的诱饵载体,并从文库中筛选ZmRop1的互作蛋白,为进一步从分子层面解析ZmRop1在玉米中的信号传导机制和提高玉米对草地贪夜蛾的抗性提供支持。【方法】以草地贪夜蛾取食后的玉米品种郑单958不同组织为材料提取总RNA,利用SMART技术反转录合成ds cDNA,连接p GADT7载体后转化到酵母Y187中构建玉米酵母双杂交c DNA文库;以玉米c DNA为模板克隆ZmRop1,构建诱饵载体pGBKT7-ZmRop1,经酶切及测序鉴定后转化到酵母AH109中,并鉴定诱饵蛋白毒性及自激活活性。通过酵母双杂交以ZmRop1为诱饵从cDNA文库筛选其潜在互作蛋白。【结果】研究获得的玉米c DNA文库库容量为4.08×10^(6)CFU,文库滴度为1.3×108CFU,文库片段多样性良好,文库重组率91.7%;诱饵载体pGBKT7-ZmRop1成功转入AH109且经过鉴定证明无毒性、无自激活活性;从文库中初步筛选到22个与ZmRop1互作的蛋白,分析表明参与调控植物防御等多种生物学活动。【结论】构建的酵母双杂交cDNA文库符合建库要求;构建的ZmRop1诱饵载体无毒性和自激活活性;筛选到22个与ZmRop1互作的靶标蛋白,为进一步研究ZmRop1参与调控植物防御的信号分子作用机制及后续筛选玉米抗虫基因打下了理论基础。

【Objective】To construct library of maize yeast cDNA induced by Spodoptera frugiperda feeding and bait vector encoding plant Rac protein,and to screen interacting protein from the library,with an aim to support further analysis on signal transduction mechanism of ZmRop1 in maize from the molecular level and improvement of maize resistance to S.frugiperda.【Method】Total RNA was extracted from different tissues of maize Zhengdan 958 fed by S.frugiperda,ds cDNA was synthesized by reverse transcription using SMART technology,and after linking to pGADT7 vector and transformed to yeast Y187,cDNA library of maize yeast two-hybrid was constructed.ZmRop1 was cloned with maize cDNA as a template to construct a bait vector pGBKT7-ZmRop1,which was transformed into yeast AH109 after enzyme digestion and sequencing identification,and the toxicity and self-activation activity of the bait protein were identified.Its potential interacting proteins were screened from the cDNA library by yeast two-hybrid with ZmRop1 as bait.【Result】The maize cDNA library obtained in this study had a capacity of 4.08×10^(6) CFU,library titer of 1.3×108 CFU,good diversity of library fragments,and library recombination rate of 91.7%.The bait vector pGBKT7-ZmRop1 was successfully trans-formed into AH109 and identified as non-toxical and non-self-activating;22 proteins interacted with ZmRop1 were initially screened from the library,the analysis showed that they involved in regulating various biological activities such as plant defense.【Conclusion】The yeast two-hybrid cDNA library constructed in this study meets the requirements for library construction;the constructed ZmRop1 bait vector has no toxicity or self-activation activity;and 22 target proteins interacting with ZmRop1 are screened,which lays theoretical foundation for further study on ZmRop1-involved signal molecule mechanism of regulating plant defense and screening of insect-resistant genes in maize.