OSHB2参与水稻铝毒胁迫响应的作用机理研究

Aluminum (Al) toxicity is the major factor limiting rice production on acidic soils, which is the research highlight that attracts plant physiologists and breeders all the time. However, the mechanism of Al tolerance in rice has not been understood completely due to comlexity of responses by Al toxicity. We speculate that OSHB2 mignt be a participant through regulating the lipid metablism based on our previous studies on genome-wide association study, genetic analysis, linkage mapping and transcriptome sequencing under aluminum stress. In this project, 1.the lipids which might act corresponding to Al stress will be studied; 2.the test about function of OSHB2 will be acted through constructing vectors of complement and CRISPR/Cas9 on OSHB2; 3.expression pattern of OSHB2 in root tip with and without Al stress and subcellular localization of OSHB2 will be studied; 4.the proteins which might interact with OSHB2 will be identified within Yeast two-hybrid system; 5.the position where OSHB2 locates among Al related genes regulatory network. The results may uncover the mechanism of action about OSHB2 corresponding to aluminum stress in rice and will supply a significant recognition about rice Al tolerance.

酸性土壤中铝毒限制水稻生产，一直是植物生理学家和育种工作者着力解决的热点问题。水稻的耐铝毒机理十分复杂，目前还未完全阐明。本研究对150份丁氏核心种质进行耐铝毒全基因组关联分析，并且构建次级F2群体进行耐铝毒遗传和连锁分析，最后从该核心种质选择耐铝和敏感品种进行转录组测序，结果发现基因OSHB2可能通过调控脂类代谢参与水稻的铝毒应答机制。本项目拟在前期工作基础上，1.鉴定参与铝毒胁迫响应的脂类；2.通过构建OSHB2的转基因互补载体和CRISPR/Cas9敲除载体对该基因进行功能验证；3.对该基因进行铝毒胁迫前后的表达模式和编码蛋白亚细胞定位研究；4.利用酵母双杂技术筛选和验证与OSHB2蛋白互作的蛋白；5.研究OSHB2在铝毒胁迫相关基因调控网络中的位置，从而揭示OSHB2的生物学功能及参与的调控网络，为更全面认识水稻的铝毒胁迫响应机制提供理论参考。

由于酸性土壤条件下的低pH值环境，使得铝以可溶性离子Al3+状态存在，致使根系遭受损伤，从而水稻生长遭受铝毒危害，最终导致水稻产量下降。水稻的耐铝毒机理十分复杂，未来仍然需要对水稻的耐铝毒机理进行深入研究，对水稻耐铝毒机制进行完善。本研究在前期发现Ⅲ类同源域亮氨酸拉链转录因子OSHB2可能通过调控脂类代谢参与水稻的铝毒应答机制。到目前为止，还没有关于该基因在水稻耐铝中作用机制的研究报道。本项目的主要研究内容为：1.对该OSHB2进行功能验证；2.鉴定参与铝毒胁迫响应的脂类；3.对该基因进行铝毒胁迫前后的表达模式和编码蛋白亚细胞定位研究；4.利用酵母双杂技术筛选和验证与OSHB2蛋白互作的蛋白。结果发现：1.通过构建互补载体和CRISPR敲除载体发现OSHB2确实参与水稻铝毒响应；2.甾醇和半乳糖脂参与水稻根尖部位的铝毒胁迫；3.OSHB2蛋白定位于细胞核；4.筛选得到8类与OSHB2互作的蛋白。通过本项目可以揭示OSHB2的生物学功能及参与的调控网络，为更全面认识水稻的铝毒胁迫响应机制提供理论参考。