OsPPR2-1调控OsGLK1控制水稻绒粘层质体发育

Tapetal proplastids develop into elaioplasts that are associated with biosynthesis of the lipids which play crucial roles in pollen wall formation in rice. It is important to study the development of proplastids in tapetal cells. In our previous studies, we showed that the down expression of cytoplasm-located PPR gene OsPPR2-1 led to the abnormal plastids in tapetal cells and pollen sterility. Transcriptomics ananlysis showed that the expression of chloroplast development related OsGLK1 and it’s down regulated genes increased in OsPPR2-1 down regulated plants. The expression of OsGLK1 was also detected in RNA immunoprecipitation of OsPPR2-1. In this project, OsGLK1 overexpression plants promoted by tapetum specific promoter and OsGLK1 RNAi plants will be obtained and the regulation net of OsGLK1 in plastids development will be observed. The regulation model of OsPPR2-1 to OsGLK1 will be identified through RNA immunoprecipitation and interaction experiment. Tapetal plastids will be observed in decreased OsGLK1 plants based on the downregulated OsPPR2-1 backgrounds. All of these will help us to understand the mechanism which OsPPR2-1 regulated OsGLK1 to control the development of plastids in rice tapetal cells.

水稻绒毡层质体发育为造油体，是花粉壁形成所必需，因此，研究绒毡层中的质体发育非常重要。在前期研究中，我们发现细胞质定位的PPR基因OsPPR2-1表达下调后，绒毡层质体发育异常，花粉育性显著下降。转录组分析表明在OsPPR2-1下调表达的植株中，调控叶绿体发育的OsGLK1及其下游调控基因的表达均显著增加，在RNA免疫共沉淀的沉降产物中可检测到OsGLK1的表达。本项目拟在此基础上，通过用绒毡层特异启动子构建OsGLK1过量表达和RNAi载体，转化水稻，研究OsGLK1对花药绒毡层质体发育的调控；通过RNA免疫共沉淀和OsPPR2-1与mRNA的互作分析，确定OsPPR2-1是如何调控OsGLK1；通过用绒毡层特异启动子干涉的方法，降低OsPPR2-1低表达植株花药绒毡层中OsGLK1的表达，观察绒毡层中质体发育的变化，进一步揭示OsPPR2-1调控OsGLK1控制绒毡层质体发育。

五肽重复序列蛋白(pentatricopeptide repeat protein, PPR)，主要定位于质体或线粒体中，在参与RNA代谢和转录后调控细胞器基因表达中发挥功能。然而，PPR蛋白是否调控细胞核编码基因的表达尚不清楚。我们的研究发现了水稻中的一个PPR蛋白，OsPPR2-1在花粉发育中的功能。与大多数其它PPR蛋白相比，OPPR2-1定位于细胞质中。下调OsPPR2-1的表达导致绒毡层细胞质体发育异常，细胞程序性死亡延长，绒毡层降解延长，花粉育性显著降低。通过转录组分析，OsPPR2-1表达下调的植株中，编码调控质体发育和维持的转录因子OsGLK1的表达显著高于野生型。此外，在RNA免疫共沉淀和RNA电泳凝胶阻滞实验的结果表明，OsPPR2-1与OsGLK1 mRNA结合。且体外切割实验进一步表明OsPPR2-1能降解OsGLK1 mRNA。值得注意的是，敲除OsGLK1基因的表达能够部分恢复OsPPR2-1基因敲除植株的花粉育性，而OsGLK1基因过表达植株也表现出与OsPPR2-1基因敲除植株相似异常的绒毡层和质体发育。综上，我们的研究表明OsPPR2-1能够通过调节OsGLK1的表达，从而控制水稻花药质体的发育和绒毡层中的PCD过程