半乳糖醛酸转移酶编码基因PDT1参与水稻育性发育的分子机理

The programmed cell death of tapetum is necessary for the development of male gamete in rice, and any abnormal in this process would result in male sterile. A nuclear male sterile mutant pdt1 was identified from EMS-treated Jinhui10 library. In pdt1, premature programmed cell death of tapetum was observed, which resulted in completely male sterility by years’ study. Genetic analysis showed that pdt1 mutant phenotype was controlled by a nuclear recessive gene. Map-based cloning and complementary verification confirmed that the PDT1 encoded a galacturonosyltransferase which is mainly expressed in panicles. Galacturonosyltransferase is a key enzyme in the synthesis process of pectin that participated in the development of tapetum. However, there is no report that the galacturonosyltransferase was invovled in male gamete development in rice. This research aims to study the function and molecular mechanism of PDT1. We propose to identify enzyme activity of pdt1 in vitro, to determine whether pdt1 mutant phenotype is related to the abnormity of pectin synthesis and metabolism of active oxygen by physicochemical and immunohistochemical study, to confirm expression pattern and protein subcellular localization of PDT1, to analysis the regulation pattern of PDT1 in transcription and protein levels, and to screen out the interacted proteins of PDT1. This study is expected to reveal the function of PDT1 and its molecular mechanism in rice fertility development.

绒毡层的程序化死亡对于水稻雄配子发育极为重要，绒毡层的异常往往会导致雄性不育。我们在水稻EMS突变体库中获得了一个完全雄性不育突变体pdt1，经多年观察鉴定，确定其绒毡层提前程序化死亡。遗传分析表明pdt1受一对隐性核基因控制。图位克隆及互补验证确定PDT1编码一个半乳糖醛酸转移酶，其在穗部表达量显著高于其它器官。半乳糖醛酸转移酶是果胶形成的关键酶，果胶参与活性氧代谢进而影响绒毡层发育，但目前并没有半乳糖醛酸转移酶参与水稻育性发育的报道。本项目拟通过对PDT1基因的功能及机理研究，鉴定pdt1突变蛋白的酶活性；探明 pdt1突变性状的理化和免疫组化缺陷，确定其是否与果胶合成及活性氧代谢异常有关；确定PDT1的表达模式和蛋白定位区域；明确PDT1在转录水平和蛋白水平的调控模式，筛选及验证其互作蛋白。本研究希望能够揭示PDT1的基因功能及半乳糖醛酸转移酶参与水稻育性发育的分子机制。

近年来，对水稻基因的克隆以及功能分析不断取得新的成果，而水稻雄性不育研究是其中的一个热点。水稻的雄性不育主要是指不能获得有育性的花粉粒，而花粉粒的发育主要在花药中完成。本研究报道了1个与雄性不育相关的水稻突变体Oryza sativa pectin defective tapetum 1（ospdt1）。相比于野生型，ospdt1突变体显示了完全不育的性状。半薄切片、透射电镜和TUNEL的结果发现ospdt1突变体绒毡层细胞的PCD进程显著提前，在8a期出现了明显的PCD信号。通过图位克隆和功能互补实验，确认LOC_Os09g36190就是OsPDT1，其编码一个半乳糖醛酸基转移酶。OsPDT1-GFP与OsPDT11-260aa-GFP在细胞内无定位信号，OsPDT11-141aa-GFP在细胞内定位于高尔基体。RT-qPCR结果显示它在水稻成熟期的根、茎、叶、鞘、穗中有表达；通过mRNA原位杂交，发现它在花药囊中的表达部位主要为雄性生殖细胞及绒毡层细胞。OsPDT1可能参与了果胶中最丰富的多糖，同型多聚半乳糖醛酸的合成。使用咔唑比色法并没有在野生型及ospdt1突变体小穗中发现明显的果胶差异。使用同型多聚半乳糖醛酸抗体JIM5和JIM7进一步检测了野生型及ospdt1突变体花药囊中的同型多聚半乳糖醛酸的分布，也没有发现明显差异。通过细胞壁连接的类受体激酶家族蛋白Oryza sativa indica Wall-associated Kinase 1（OsiWAK1）的定位结果，发现ospdt1的胞内果胶可能产生了缺陷，影响了OsiWAK1-EYFP的胞内分布。OsiWAK1的敲减植株（WI）进行育性检测，发现它的育性严重下降，其绒毡层细胞的PCD进程提前。OsiWAK1的超表达植株（WEO）育性也严重下降，并且在花药发育的成熟期出现了四分体样的花粉粒，其花药绒毡层细胞的PCD进程延迟。ospdt1/WEO部分恢复了ospdt1的表型。综上所述，OsPDT1的突变可能引起细胞内果胶的缺陷，从而影响OsiWAK1的细胞内定位及功能，并造成绒毡层PCD异常与完全的雄性不育性状。