高盐胁迫下水稻幼苗形成关键基因qSE3克隆与功能分析

It is a valuable way to solve the rice salinity damage by breeding salt-tolerant rice varieties with good seed vigour though understanding the molecular mechanism of rice seedling establishment under high salinity stress. In our previous study, a novel QTL qSE3 for seedling establishment located on rice chromosome 3 were identified under 300 mM NaCl condition in the salt-tolerant japonica landrace Jiucaiqing. It was mapped in the physical interval of 9.921 kb, and the candidate gene ion transporter was preliminarily confirmed in this region. In this study, the mutants of qSE3 created with CRISPR/Cas9 and T-DNA insert approach and the qSE3 overexpression transgenic lines obtained with Agrobacterium-mediated approach will be used to identify the function of qSE3. Through physiological, biochemical as well as molecular and bioinformatic techniques, it will be explored the possible physiological and molecular mechanism of qSE3 in promoting rice seed germination and seedling establishemnt. Meanwhile with more than 400 rice germplasms sequenced it will be identified the genetic variation and evolution of qSE3 and the polymorphism of qSE3 allele in order to develop the SNP haplotype of qSE3. The practical molecular markers of qSE3 will be developed using SNP haplotype, combined with high-density molecular linkage in the region of qSE3. The results will serve as the genetic improvement in salt tolerance of rice varieties through molecular breeding technology in the future.

揭示盐胁迫下水稻种子萌发和幼苗形成的分子遗传机理，选育和利用高种子活力耐盐水稻品种是解决水稻盐害最经济有效的方法。课题组已经在强耐盐粳稻地方品种韭菜青中检测到一个新的控制盐（300 mM NaCl）胁迫的幼苗形成关键基因qSE3，定位于水稻第3染色体9.921kb物理区间，初步确定是一个离子转运蛋白基因。本项目拟利用CRISPR/Cas9基因敲除技术获得候选基因的点突变体、购买获得候选基因的T-DNA插入突变体及利用农杆菌介导法获得的基因过量表达转基因植株验证基因功能；通过生理生化分析、基因表达分析及基因参与的信号通路分析，探讨qSE3候选基因作用的生理生化和分子机理；利用400份不同来源水稻资源的测序信息，进一步研究qSE3基因遗传多样性及进化，发掘目标基因单倍型SNP位点，开发与其紧密连锁的、实用的分子标记，为利用生物技术培育高种子活力耐盐水稻品种提供基础。

揭示盐胁迫下水稻种子萌发和幼苗形成的分子遗传机理，选育和利用高种子活力耐盐水稻品种是解决水稻盐害最经济有效的方法。课题组已经在强耐盐粳稻地方品种韭菜青中检测到一个新的控制盐（300 mM NaCl）胁迫的幼苗形成关键基因qSE3，定位于水稻第3染色体9.921kb物理区间，初步确定是一个钾离子转运蛋白基因OsHAK21。本项目进一步通过亲本间基因组序列比较和转基因验证，明确了qSE3目标候选基因为编码钾离子转运体基因OsHAK21基因。组织表达显示，OsHAK21在种子发育和盐胁迫下种子萌发过程的中有重要作用。同时，OsHAK21调控盐胁迫下种子萌发中钠钾离子含量、ABA和GA含量与相关基因表达和抗氧化活性氧系统（ROS）。转录组结果显示，qSE3涉及非生物胁迫响应代谢。表明，该基因可能通过影响钾离子稳态、植物激素ABA和GA途径与ROS途径调控盐胁迫下种子萌发和幼苗形成。另外，通过OsHAK21单倍型分析，测定到单倍型HAP3是OsHAK21基因一种优异单倍型，能够显著性提高盐胁迫下萌发与成苗能力。直播模拟试验，显示qSE3基因在盐胁迫下种子萌发幼苗形成中具有育种利用价值，同时获得具有应用价值的盐胁迫下高活力材料2份，这将有助于培育高活力水稻育种品种。