橡胶树草甘膦抗性关键基因HbEPSPS的结构与功能解析

Rubber tree seedlings are subject to phytotoxicity of glyphosate, which leads to the leaf falling, distorted new leaf phenotypes, etc. 5-enolpyruvoylshikimate-3-phosphate synthase gene (EPSPS) is target site for genetic improvement for glyphosate-resistant GM crops and a key gene for solve phytotoxicity of glyphosate. We had proved physiological response of phytotoxicity of glyphosate leading to rubber tree leaves distortion. Recently, we found HbEPSPS gene expression was related to phytotoxicity of glyphosate in rubber tree, and upregulated by ABA and ZT. Study the structure and function of HbEPSPS gene is effective way to solve scientific problem of phytotoxicity of glyphosate in rubber tree. In this study, changes of physiology, hormones and transcriptome after phytotoxicity of glyphosate in rubber tree seedling leaves will analyze by means of molecular biological techniques; analyze HbEPSPS gene expression patterns in different leaves stages of phytotoxicity of glyphosate; select HbEPSPS glyphosate resistance gene by means of Site-directed mutagenesis PCR technology, and prove its function in transgenic Arabidopsis. This study will clarify the damage mechanisms of HbEPSPS on rubber tree, clarify the relationship between structure of HbEPSPS and function of glyphosate resistance, and provide theoretical basis for glyphosate resistance research and practical guidance for glyphosate resistance varieties breeding in rubber tree.

橡胶树幼苗受到草甘膦药害后，导致叶片脱落、新生叶片扭曲等表形。5-烯醇式丙酮酸莽草酸-3-磷酸合成酶基因（EPSPS）是抗草甘膦除草剂转基因作物遗传改良的主要位点，是解决橡胶树草甘膦药害的关键基因。最近，在发现橡胶树草甘膦导致叶片变形的生理响应的基础上，我们证明HbEPSPS基因表达与橡胶树草甘膦药害有关，并受脱落酸和玉米素诱导上调表达。研究HbEPSPS结构与功能是解决橡胶树草甘膦抗性这一科学问题的有效途径。本研究采用分子生物学技术，检测橡胶树苗期叶片草甘膦药害后中生理、激素和转录水平的变化规律；分析HbEPSPS基因在橡胶树草甘膦药害不同时期叶片的表达规律；采用定点诱变PCR技术筛选HbEPSPS草甘膦抗性基因，并转入拟南芥验证其功能。本研究将阐明草甘膦对橡胶树生长的危害机制，阐明HbEPSPS的结构与橡胶树草甘膦抗性的关系，为橡胶树草甘膦抗性研究和培育橡胶树草甘膦抗性品提供理论基础。

针对橡胶树种植胶园喷施草甘膦除草导致橡胶树幼苗发生药害的现象。我们提出5-烯醇式丙酮酸莽草酸-3-磷酸合成酶基因（EPSPS）是抗草甘膦除草剂转基因作物遗传改良的主要位点，是解决橡胶树草甘膦药害的关键基因这一科学问题。本项目首先确定橡胶树幼苗受到草甘膦药害后导致叶片脱落、新生叶片扭曲等表型变化的浓度梯度，分析草甘膦药害对橡胶树叶片光合作用、抗氧化物酶活性和植物激素响应基础，发现其导致ABA含量升高。进一步从橡胶树叶片中克隆了HbEPSPS基因，证明其在叶片、树皮等组织中组成型表达，在叶片中受ABA，JA和IAA等激素诱导显著上调表达。采用草甘膦处理后测定了0-3d的转录组，预测59,608个CDS，预测转录因子编码基因2,389个。采用生理生化方法，创新性的发现ABA可有效缓解草甘膦对橡胶树芽接苗的药害症状。据此，鉴定了橡胶树ABA信号途径6个HbSnRK2，95个PP2C和85个bZIP基因家族成员，证明ABA信号转导基因家族成员受草甘膦诱导差异表达。发现HbEPSPS启动子区域具有bZIP结合位点。采用点突变技术获得四个点突变位点GLY95B，GLY101C，THR182D和ALA267T。并证明过表达GLY101C和THR182D点突变转基因拟南芥对草甘膦抗性显著提高。本研究证明ABA缓解草甘膦药害的作用，草甘膦对橡胶树生长的危害与HbEPSPS直接相关，HbEPSPS的GLY101C和THR182D点突变可提高草甘膦抗性。本项目为橡胶树草甘膦抗性机制研究和培育橡胶树草甘膦抗性品提供理论基础和技术指导。