小麦TaVIP2基因的克隆、功能鉴定及分子机理研究

Wheat is considered to be the world’s most important crop, but its genetic engineering breeding has lagged behind the other major grain crops because of the inefficient transformation technique. It might be a vital strategy to manipulate the expression of some host proteins involving in the integration of T-DNA inside wheat cells for the enhancement of stable transformation efficiency. In our previous study, a TaVIP2 gene which encodes a VirE2-interacting protein (VIP) was cloned in common wheat. By heterologous expression in tobacco, TaVIP2 gene was identified to improve the efficiency of Agrobacterium-mediated transformation and have resistance to powdery mildew. In this applying project, full length sequences of all TaVIP2 genes will be isolated in common wheat and its three ancestors. Then, their molecular structures will be investigated and the evolutionary relationship between wheat and theirs ancestors will be analyzed. Further, their biological functions will be ascertained by transformation strategy, resistance assays for biotic or abiotic stress and phenotypic analysis. Moreover, by studying the transgenic wheat plants combining with qRT-PCR, yeast two-hybrid, transcriptomics and bioinformatics methods, the molecular mechanism of TaVIP2 gene in T-DNA integration process will be analyzed. Our output will provide theoretical basis for well understanding the molecular mechanism of TaVIP2 gene in the T-DNA delivery process of Agrobacterium-mediated strategy, and also will be very potential for the efficiency improvement of Agrobacerium-mediated transformation of wheat.

低效率的遗传转化技术导致小麦基因工程育种远远落后于其它主要粮食作物，通过调控小麦中与T-DNA整合相关基因的表达是提高T-DNA整合效率和小麦稳定转化效率的关键。我们已从小麦中克隆到一个与VirE2互作的TaVIP2基因，在烟草中的功能鉴定表明该基因可以提高农杆菌稳定转化效率，并首次发现其还具有抗白粉病效果。本项目拟进一步从小麦及其祖先种中克隆TaVIP2的全长序列，探讨TaVIP2基因间的分子结构特征，分析小麦与祖先种基因组间的进化关系；同时综合利用转基因、抗逆性鉴定及表型分析等方法，明确TaVIP2基因的生物学功能；进一步以过表达TaVIP2的转基因小麦为材料，结合qRT-PCR、酵母双杂交、转录组学和生物信息学等方法，解析TaVIP2在T-DNA整合过程中的分子机理。该项研究对深入认识和阐明TaVIP2基因在农杆菌转化植物过程中的调控机理，提高小麦农杆菌转化效率具有重要意义。

一直以来小麦是一个难转化的物种。其中将与T-DNA整合过程相关的一些宿主基因的表达进行干预可能是解决这一难题的一种潜在方法。VIP2蛋白在T-DNA转运和整合过程中起着重要的作用。利用同源序列克隆技术，以拟南芥AtVIP2基因编码序列作为种子序列，从普通小麦中克隆了TaVIP2基因，测序结果表明该基因1839bp，与拟南芥AtVIP2蛋白质序列相似性为48%。Southern blotting杂交结果表明，TaVIP2在小麦基因组中有3个拷贝。进一步以硬粒小麦与粗山羊草代换系为材料，利用Southern blotting技术将TaVIP2定位在小麦1A、1B和1D染色体上。分别从小麦AA、DD、SS野生近缘种中扩增出了3个TaVIP2基因的cDNA和gDNA，发现TaVIP2基因组序列含有13个外显子和12个内含子。亚细胞定位分析表明，TaVIP2蛋白定位于细胞膜、细胞质和细胞核。酵母双杂交结果发现，TaVIP2蛋白与农杆菌VirE2蛋白特异性互作。利用农杆菌介导法将小麦TaVIP2基因转入烟草和小麦中，发现过表达小麦TaVIP2基因能将农杆菌介导的烟草的转化效率提高2.5倍以上，同时发现转基因烟草具有白粉病抗性。此外，利用实时定量PCR对白粉病抗性相关基因在转基因烟草中的表达进行了分析，研究发现内源基因NtPR-1的表达量有明显上调，推测该基因可能与转基因植株的白粉病抗性有关。该研究进一步明确了TaVIP2促进T-DNA整合从而提高农杆菌介导的烟草和小麦的转化效率，并利用该基因的研究结果建立了稳定高效的农杆菌介导的小麦遗传转化体系