天冬氨酸蛋白酶OsAP17和OsAP18调控水稻花粉萌发的分子机制研究

As a crucial prerequisite for seed formation in plant, male gametogenesis is extremely essential for rice fertility. Recent studies have identified a diverse range of factors and their related pathways in the early formation of male gametophyte. However, molecular mechanisms in late process of male gametogenesis such as pollen germination are poorly understood. By functional analysis in the complete set of aspartic protease (AP) gene family in rice, the applicant has identified that knockout of either OsAP17 or OsAP18, both of which encode an aspartic protease (AP) in rice, results in abnormality in pollen germination. These two APs show exactly the same phenotype, although they have distant phylogenetic relationship. Reciprocal crosses between the wild type and heterozygous mutants of the two APs demonstrated that the mutant alleles could not be transmitted into the next generation through the male gametophyte. Considering the preferential expression of the two APs in anther, these two genes might play important roles in pollen germination. .Based on these results, we plan to understand the function of the two APs in three aspects. First, we plan to investigate a set of characters in genetic materials with different genotypes, including the ultrastructure of the extine, the viability and germination of the pollen grains, the elongation of the pollen tube and so on. Combined with the expression analysis and the subcellular localization of the APs in the process of pollen germination, the molecular functions can be inferred. Second, the function of a gene is determined by its structure. Thus we would like to explain that whether the same phenotype is determined by the homologous AP domain or by other sequences in these two APs, in the light of evolution. Furthermore, the PCD-related genes and genes involved in carbohydrate metabolic process were found to be up- or down-regulated in the mutants, which showed the possible mechanisms in regulating pollen germination by APs. Therefore, we plan to investigate the differentially expressed genes in the transgenic plants by microarrays, in order to identify the pathway-related genes of the two APs. The interacting proteins of OsAP17 and OsAP18 will be identified by yeast two-hybrid. The Co-IP and BiFC assay will be used for confirmation of the interaction. Taken together, our research will provide direct evidence in understanding whether APs are required for pollen germination by conserved or divergent mechanisms.

雄配子发育决定植物能否产生正常后代，对水稻结实率有直接影响。迄今为止对水稻雄配子的研究主要涉及早期发育阶段，而对花粉萌发等发育后期的研究还有待深入。申请人在研究天冬氨酸蛋白酶AP家族基因功能时，发现进化上属不同分支的两个AP突变体表型相同，插入缺失后花粉萌发异常，插入T-DNA的AP基因不能通过雄配子传递到后代。两个AP在雄蕊高表达，推测其功能都和花粉萌发相关。.本项目1.拟从不同遗传材料的花粉外壁结构，花粉活力和萌发，花粉管生长等表型入手，结合表达分析和两个AP在花粉萌发中的亚细胞定位研究其功能。2.鉴于两个AP的序列特征，探究其功能是由高度同源的AP结构域还是其他序列所决定，从进化的角度研究两个AP序列和功能的关系。3.突变体中糖代谢和PCD基因显著上调或下调，在此基础上进一步检测转基因植株的差异表达基因，并分离与之直接作用的蛋白，最终回答两个AP是否通过类似或不同的机制影响花粉萌发。

天冬氨酸蛋白酶（AP）是四大水解酶之一，广泛存在于动物、微生物、病毒和植物中。水稻基因组至少编码96个天冬氨酸蛋白酶，目前只对其中少数几个进行了功能研究。本研究通过筛选水稻中AP基因的突变体，获得了一个控制花粉萌发和花粉管发育的天冬氨酸蛋白酶基因，该基因在水稻OsAP家族中被命名为OsAP65。OsAP65突变体后代出现严重的偏分离，T-DNA插入杂合植株后代只有野生型和杂合型，没有纯合突变型植株，且分离比约为1:1。通过杂合植株和野生型植株的正反交实验表明，没有T-DNA插入纯合个体是由于雄配子不能正常传递造成的，对杂合植株的花粉进行细胞学观察，发现突变型花粉和野生型一样，能够发育成熟。花粉体外萌发实验表明，杂合植株花粉管萌发率低于野生型。利用Real-time PCR对OsAP65的表达进行检测，发现OsAP65在全生育期都有表达，并且在杂合植株中的表达量是野生型中的一半。对OsAP65进行亚细胞定位，结果表明OsAP65主要定位在PVC。通过功能互补实验表明OsAP65可以互补偏分离表型。这些结果表明，OsAP65参与了水稻花粉管的发育过程。. 为了进一步研究水稻花粉萌发和花粉管伸长的生理生化机制，选用包括不同籼稻和粳稻在内的12个水稻品种，完成17个不同组合的杂交实验，检测不同水稻材料杂交后，花粉在柱头的附着、萌发和花粉管的伸长。通过扫描电子显微镜观察花粉在柱头上的附着，发现2/3的亚种间杂交组合柱头平均附着花粉粒数不足5粒；3/4的亚种内杂交组合附着花粉粒数多于5粒。同时观察到亚种间杂交花粉管伸长多出现空瘪、盘绕等异常现象，亚种内水稻品种的组合比较正常。花粉萌发和花粉管的正常发育决定植物能否产生后代，对水稻结实率有直接影响。研究结果有助于进一步理解水稻花粉萌发和花粉管伸长的机制，同时对提高水稻产量有一定启发意义。