miR1432调节水稻籽粒灌浆充实的作用机制

Grain weight and quality are always determined by the grain filling. It has been proved that miR1432 have important roles in regulating rice grain filling. Compare with wild-type plant, the brown rice weight of STTM1432 transgenic lines (miR1432 lower expressed) was significantly increased. In contrast, the brown rice weight of MIR1432 transgenic lines (miR1432 higher expressed) was significantly decreased. In this project, 5’-RACE was employed to identify the real target of miR1432.Over-expression and down-expression of miR1432’ target transgenic lines will be generated in rice use endosperm specifically expressed promoter, and the grain filling rate will be investigated in these transgenic lines to detect the downstream target of miR1432 in controlling rice grain filling. Then, endogenous hormone, sucrose and starch metabolism related genes’ expression in endosperm of these transgenic lines will be quantified to study the molecular mechanism of miR1432 in controlling rice grain filling. After that, endosperm cell proliferation, seed size, endogenous hormone content, sucrose and starch metabolism related enzyme activity will be assayed to study the physiological mechanism of miR1432 in controlling rice grain filling. By explaining the mechanism of how miR1432 regulate rice grain filling, it will be helpful to provide theoretical guide for rice yield improvement by means of molecular breeding.

水稻籽粒灌浆充实的好坏决定了稻米产量的高低及其品质的优劣。项目申请人前期研究已证明，在胚乳中低表达miR1432显著增加转基因株系糙米的千粒重，而高表达miR1432显著降低转基因株系糙米的千粒重。本项目拟在上述研究基础上，采用5’-RACE鉴定预测的miR1432靶基因，并利用转基因技术在胚乳中高表达和低表达miR1432的靶基因，互补miR1432低表达和高表达的表型，结合miR1432的时空表达特性、各转基因株系籽粒灌浆过程中內源激素、蔗糖-淀粉合成代谢相关基因的表达，解析miR1432调节水稻籽粒灌浆充实的调控网络和分子机制；进一步通过测定各转基因株系胚乳细胞的分化和增值过程、籽粒的灌浆速率和大小，籽粒灌浆过程中內源激素含量、蔗糖-淀粉代谢关键酶活性，分析miR1432调节水稻籽粒灌浆充实的生理机制，为利用分子改良育种改善水稻籽粒的灌浆充实，提高水稻的产量和品质提供技术支撑。

水稻籽粒灌浆充实对水稻产量的形成和营养物质的积累具有重大影响。MiRNA是植物生长发育的重要调控因子，对植物生理进程具有重要调控作用。本项目发现通过短片段靶基因模拟物（STTM）技术抑制水稻miR1432（STTM1432）的表达可以显著提高水稻籽粒的灌浆速率，从而使水稻籽粒的千粒重得到显著提高。大田实验研究结果发现，低表达miR1432使水稻产量提高17.14%。分子试验证据表明，miR1432通过切割的方式负调控其下游靶基因OsACOT的表达。通过同源分析发现OsACOT是一个编码乙酰硫脂酶蛋白（Acyl-CoA thioesterase）的基因，且在其他物种中具有较高的保守性。更重要的是，过量表达抗miR1432切割版本的OsACOT (OXmACOT)可以很好的互补STTM1432转基因植株的表型，且OXmACOT转基因株系通过促进灌浆充实使籽粒的千粒重比野生型提高46.69%。进一步研究结果表明，OsACOT参与中链脂肪酸的合成过程。另外，通过对miR1432转基因株系RNA-seq数据进行分析发现，miR1432通过调控下游基因进而参与脂肪酸代谢和植物激素合成等过程，这与OsACOT共表达基因的富集结果一致，并与STTM1432及OXmACOT转基因植株中生长素和脱落酸含量的升高相吻合。综上，miR1432-OsACOT调控模块在水稻籽粒灌浆及作物产量提高方面具有重要作用。相关研究结果发表于Molecular Plant、Plant Biotechnology Journal等国内外著名学术期刊。