miR528参与调控水稻砷耐性的分子机理研究

The production and quality of rice as well as human health are severely challenged by environmental arsenic exposure. Growing evidence shows that one of the effective ways to resolve rice arsenic exposure is to enhance the rice plants ability to arsenic tolerance. However, only a few of protein-coding genes with potential relevance with arsenic tolerance have been identified in rice so far. In our previous studies, we found that transgenic plants containing the over-expression miR528 vector become significantly more sensitive to arsenite than the wide-type japonica rice Nipponbare that is tolerant to arsenite, suggesting that this gene may participate in the processes of arsenic responses in rice. In this project, taking the rice arsenic pollution and miRNA gene studies into consideration, we want to further uncover the biological function of miR528 in regulation of rice arsenite tolerance and the underlying molecular mechanisms on the basis of our previous results, using the combination of approaches of RACE, RNA-Seq high-throughput sequencing, GUS reporter gene fusion, gene transformation, and SRXRF technology, etc. To this end, a construct expressing the target mimicry MIM528 will have been constructed firstly. Using the functional complementary experiments, we want to clearly clarify the relationships between miR528 and arsenite tolerance by comparing the phenotypes of transgenic plants over-expressing miR528 (OE-miR528) and MIM528 (OE-MIM528) under arsenite stress. Secondly, we want to have identified one or several miR528 target genes that play most important roles in regulation of rice arsenite tolerance (termed "critical targets"), by combinations of in silico prediction, RACE validation, target genes transformation with either the normal or engineered coding sequences, as well as hybridization with OE-miR528 plants, etc. Thirdly, the temporal and spatial expression patterns of miR528 and its critical target genes will have been in situ explored using the fusion of GUS reporter gene. Lastly, using the transgenic and wild-type rice plants as materials, the microstructure of root and stem cells will have been photographed and compared, as well the distribution of arsenic and copper in different tissues, when the plants are cultivated under normal circumstance and arsenite stress, respectively. In addition, the physiological characteristic and ability in arsenic efflux of different genotypes will have been investigated and compared. Taken together, we attempt to disclose the molecular mechanisms of arsenic tolerance mediated by miR528 and its critical targets in rice. The results expected would deepen our knowledge in further understanding the interactions between rice and arsenic. On the other hand, it would be informative and significative for stress-resistant crop breeding or improvement in rice by supplying a mount of novel gene resources.

砷污染严重影响水稻的产量、品质和人类健康。提高水稻的砷耐受性是解决水稻砷污染有效途径之一。但目前仅有少数基因被发现或与水稻的砷耐性有关。我们发现，在耐砷品种日本晴中过表达miR528后可使其砷耐性极显著下降。项目瞄准水稻砷污染和miRNA研究热点，拟在前期研究基础上，综合运用RACE、RNA-Seq测序、启动子融合GUS、转基因、SRXRF技术等，经过表达和抑制表达miR528，探明其与水稻砷耐性的关系；通过生物信息预测、RACE验证、miR528敏感和不敏感的靶基因过表达转基因及与miR528过表达植株杂交等，鉴定出在砷胁迫下受到miR528调控的主作用靶基因；原位解析miR528和主作用靶基因作用的组织部位和时期；明晰由其介导水稻砷耐受的细胞学基础和生理特征，以此阐明miR528与主作用靶基因调控水稻砷耐性的作用机理，从而为加深水稻-砷互作关系认识及水稻抗逆育种提供新思路和新靶标基因。

水稻砷污染是世界性环境与卫生重大问题，迫切需要解决。本项目利用过表达和抑制表达转基因技术发现，miR528负向调控水稻的砷耐受性；该基因的过表达转基因水稻对砷高度敏感，与野生型日本晴（WT）相比，其根系的砷吸收能力下降但向地上部转移能力增加，而抑制该基因的表达后其砷耐受性却与WT相当。采用生物信息学预测结合定量RT-PCR、RNA-Seq测序、烟草叶片瞬时转染等技术手段，初步确定了miR528的8个候选靶基因；进一步对这些靶基因进行过表达转基因及砷处理下的生理指标分析等，明确OsCBP1应是miR528调控水稻砷耐性的主作用靶基因。利用定量RT-PCR、启动子融合GUS和转基因等，发现miR528为广谱性表达，在水稻根系、茎干、叶片、雄蕊和颖壳等部位均有表达；OsCBP1在胚、胚乳和根系等组织器官中表达量较高。石蜡切片分析发现，miR528被过量表达后其茎干中维管束的数量明显增加。对各生理指标分析发现，WT和各转基因材料间SOD等抗氧化酶活性、氨基酸含量等均存在显著差异，而且miR528过表达转基因水稻根系的砷外排能力显著低于WT。另外，miR528过量表达可致使水稻株高矮化和分蘖数增加。这些结果为深入阐明水稻应答砷胁迫的作用机理奠定了坚实基础，并为水稻耐逆育种或品种改良提供了参考依据和重要基因资源。