水稻砷污染是世界性重大问题亟需解决。提高水稻的砷耐性，减少砷积累是解决该问题的有效途径。但目前仅有少数基因被发现与水稻的砷积累和耐性有关。我们发现，OsRAV1是MIR528的上游调控因子，两者的过表达和功能敲除突变体具有截然相反的砷耐受表现。项目拟在前期工作基础上，利用qRT-PCR、启动子融合GUS和转基因等，明晰两个基因是否存在共表达模式；运用ChIP-Seq、EMSA、原生质体转染、过表达和敲除突变体间杂交等，搞清OsRAV1调控MIR528表达的分子机制及两者间的遗传互作关系；利用转录组和ChIP测序的关联分析及敲除表达转基因等，挖掘OsRAV1的其它主效下游基因并弄清它们在砷胁迫应答中的功能；分析和比较转基因水稻砷吸收、外排、转运与积累差异及生理指标变化等，探明OsRAV1-MIR528调控水稻砷积累与耐性的内在机理，以加深对水稻—砷作用关系认识并为水稻耐逆育种提供新靶标基因。

Arsenic (As) contamination in rice is a critical issue worldwide that needs to be figured out urgently. It is demonstrated that improving As tolerance and reducing As accumulation are effective ways to solve the problem of As contamination in rice. However, only a few of genes have been evidenced to be involved in As accumulation and tolerance in rice so far. In our previous study, we found that OsRAV1 can bind onto the promoter of MIR528, and transgenic rice plants separately carrying overexpressed and repressed OsRAV1 or MIR528 gene showed distinct As accumulation and tolerance under arsenite (AsIII) stress, suggesting that these two genes should participate in the process of As response in rice. In this research project, taking the rice As contamination into consideration, we want to further uncover the molecular mechanism of OsRAV1-MIR528 interaction in regulation of As accumulation and tolerance in rice on the basis of our previous findings. To this end, the temporal-spatial expression pattern of OsRAV1 and miR528 in different tissues and developmental stages will be firstly explored to examine whether the two genes were co-expressed, using real-time RT-PCR, transformation of constructs separately expressing OsRAV1 or MIR528 promoter in fusion of GUS reporter gene, and histochemical staining. Secondly, we intend to uncover the molecular mechanism of regulation of MIR528 expression by OsRAV1 as well as the genetic interaction relationship between OsRAV1 and MIR528, via chromatin immunoprecipitation assay sequencing (ChIP-Seq), electrophoretic mobility shift assay (EMSA), rice protoplast transfection, and analysis of phenotypes of hybrids between mutants with overexpressed or repressed OsRAV1 and MIR528 genes under AsIII stress etc. Thirdly, we want to identify other major downstream target genes of OsRAV1 by using approach of combination of analyses of RNA-Seq and ChIP-Seq data, and further investigate their biological function in response to AsIII stress by creating rice mutants with target genes knocked out using Crispr/Cas9. Lastly, using WT and transgenic rice plants as materials, the difference of ability in As uptake, efflux, translocation and accumulation as well as the changes of physiological and biochemical indexes of different genotypes will be examined and compared to further understand their physiological characteristic. Taken together, we will finally disclose the molecular mechanism of As accumulation and tolerance mediated by OsRAV1-MIR528 interaction in rice. The results expected would deepen our knowledge in further understanding the relationship between rice and As. On the other hand, it would be informative and significative for stress-tolerant crop breeding by supplying for novel protein-coding gene(s) in rice.