小麦低温响应蛋白PAP6-like互作蛋白筛选及功能分析

低温尤其是倒春寒是影响小麦产量的主要非生物胁迫因子之一，近年来造成我国小麦严重减产。申请人前期利用一个小麦重组近交系群体和蛋白组学技术，发现了一个耐寒性关键基因PAP6-like；病毒诱导基因沉默（VIGS）和EMS突变体结果表明，该基因在增强小麦低温包括倒春寒抗性方面发挥重要作用。在此基础上，本研究以PAP6-like蛋白为诱饵，利用酵母双杂技术对已构建的小麦酵母双杂文库进行互作蛋白筛选，进一步通过双分子荧光互补和免疫共沉淀技术对关键候选蛋白与PAP6-like的互作关系进行验证；并利用VIGS技术、小麦EMS突变体、CRISPR/Cas9技术以及超表达转基因小麦对其中1个关键候选基因的低温抗性包括倒春寒响应功能进行验证。本研究能够为我国小麦抗逆育种提供重要基因资源，同时也将有助于进一步解析小麦低温响应的遗传基础和调控机理。

Cold stress especially spring frost is one of the major abiotic stresses that effects wheat yield, and also an important factor causing wheat yield reductions in China. One key cold tolerant gene PAP6-like was identified using a recombinant inbred line (RIL) population and proteomics technique in our previous study. Virus-induced gene silencing (VIGS) and tetraploid wheat ethyl methane sulfonate (EMS) mutant indicate that this gene plays a vital role in conferring cold including spring frost tolerance in wheat. On this basis, this research plans to utilize PAP6-like as bait to screen the interactive proteins in built wheat yeast library using yeast two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) will be used to further validate the protein interaction between key candidate proteins and PAP6-like. The responsive function of one key candidate gene involved in cold including spring frost stress will be verified by VIGS, wheat EMS mutants, CRISPR/Cas9 technology and transgenic wheat with overexpression. This study will not only provide valuable genetic resources for wheat resistant breeding in China, but also be helpful to the further research of the genetic basis and regulatory mechanism for wheat cold response.