水稻氧化胁迫响应基因OsSRT的功能研究

Wherever and whenever plants are frequently exposed to adverse unfavorable biotic and abiotic stresses, oxidative stress occured and accompanied as well, plant growth, development, and productivity can be seriously affected by stress. Although the currently progress has revealed many physiological pathways responded to oxidative stress in plants, but histone deacetylase ( Surtuin ) involved in the plants interacted with oxidative stress have not been reported as an important post-translated regulatory protein. Our preliminary data showed that two OsSRTs from rice surtuin family were strongly induced under oxidative stress. By using molecular genetics, physiology and biochemistry assays, the project would emphasize to construct RNAi mutants of OsSRT gene, and to construct OE mutant of OsSRT gene, and to identify phenotype of OsSRT mutants, to analyze the relatives of OsSRT with the related genes responded to oxidative stress, to clarify the rules on metabolic pathways; on the other hand, the recombined protein of OsSRT would to be constructed in vitro, to obtain purified OsSRT protein with high activity, and to identify enzymatic function of target protein. The expected results would illustrate the function of OsSRT, explore the role of OsSRT in cell metabolism and growth, reveal the plant molecular mechanism of target genes involved and regulated to oxidative stress response, and also provide new gene resources to improve the genetic improvement of plant tolerance.

植物遭受各种生物及非生物胁迫时，氧化胁迫也普便发生，严重影响植物的生长与发育。目前的研究进展尽管已揭示了多条植物应对氧化胁迫的生理途径，但作为一类重要的蛋白质修饰调控分子- - 组蛋白去乙酰化酶（Surtuin）参与植物氧化胁迫的生理活动未见报道。我们的前期研究发现，水稻中Surtuin家族的2个成员OsSRT分别被氧化胁迫强烈诱导表达。本项目采用分子遗传学、生理生化等手段，创制水稻OsSRT基因的抑制突变体、超表达突变体，鉴定OsSRT基因的突变体表型，分析OsSRT与其他氧化胁迫相关基因的关系，明确细胞代谢途径的变化规律；体外重组OsSRT蛋白，获得纯化的OsSRT活性蛋白，鉴定其酶学功能。预期结果将明确水稻OsSRT蛋白的功能，探讨OsSRT基因在细胞代谢与生长中的作用，揭示该基因参与植物对氧化胁迫响应调控的分子机制，也为提高植物抗性的遗传改良提供新基因资源。

在氧化胁迫下，植物组蛋白去乙酰化酶（Surtuin）调控细胞进行响应。我们的研究发现，水稻中Surtuin家族的成员OsSRT被氧化胁迫强烈诱导表达，同时OsSRT基因的上调以及由氧化胁迫诱导出的H2O2互作调控细胞的代谢。本研究结果显示，水稻ossrt1，ossrt2，ossrt1/2突变体分别对氧化胁迫表现为高度敏感，OsSRT基因的表达降低，表型生长异常，出现褐化斑点、苗期衰亡、植株矮小、生殖发育障碍，鲜重、根长及叶绿素含量下降，细胞的H2O2含量上升；超表达35S:OsSRT1/2对氧化胁迫表现为高抗， APX、POD、CAT等抗氧化酶基因表达上调，细胞的H2O2代谢平衡，表型与野生型没有差异。OsSRT基因在水稻各器官中均有表达，尤其根尖和茎尖中表达量最高，表达量与时间、氧化胁迫强度成正比关系；OsSRT启动子核心区域序列的活性受氧化胁迫诱导，确定了与氧化胁迫响应存在增强作用的顺式调控元件。体外OsSRT融合蛋白的研究结果表明，优化了对His-OsSRT融合蛋白在体外体系进行的诱导表达，确定了IPTG用量、诱导时间、温度等关键因素；获得了高度纯化的OsSRT蛋白，确定了OsSRT催化特性的专一性是对组蛋白H3K9的去乙酰化。从以上数据结果推断，水稻在氧化胁迫下诱导了体内活性氧的产生，伴随引发了OsSRT的修饰调控，以及由二者共或互作启动了下游相关抗氧化基因的表达响应，从而调控了基因的表达，修饰了胁迫下的蛋白质，维持了细胞稳定所需的代谢需求。上述结果不仅进一步阐明水稻OsSRT蛋白在氧化胁迫下的表现与功能，探讨OsSRT基因在细胞代谢与生长中的作用;最终不仅为揭示该基因参与植物对氧化胁迫响应调控的分子机制提供理论依据，也为提高植物抗性的遗传改良提供新基因资源。发表SCI论文2篇、EI论文1篇、核心期刊论文2篇，出版学术专著1部，培养硕士研究生8名。