水稻PPR5蛋白突变引起粉质胚乳表型的发育机制

Rice floury endosperm mutants are important genetic materials for analyzing endosperm development and its regulation mechanisms. In previous research, we isolated a new rice floury endosperm mutant ppr5 (pentatricopeptide repeat 5). The protein contents in ppr5 endosperm significantly decreased and starch components changed. PPR5 is located in mitochondria and participates in the post-transcriptional process of mitochondrial genes. However, the development mechanism of floury endosperm caused by PPR5 mutation is still unclear. In this project, developing endosperms of mutant ppr5 and its wild type will be used as plant materials. Firstly, the nutritional transport tissue, construction and differentiation process, and ultrastructure (especially mitochondria) of endosperm will be observed with morphology analysis techniques to reveal the effects of PPR5 mutation on kernel development. Secondly, the transcription products and intron splicing efficiency of endosperm mitochondrial genes will be analyzed, the abundance and activity of mitochondrial respiratory chain complexes will be determined, and the ATP content in endosperm will be measured through physiological, biochemical and molecular biological methods. These studies will clarify the mechanism of PPR5 involved in mitochondrial structure and function. Thirdly, the metabolic pathway of PPR5 involved in starch and protein synthesis, development or accumulation in endosperm will be investigated through RNA-seq and molecular biology techniques. The results will elucidate the development mechanism of rice floury endosperm caused by PPR5 mutation, expand acknowledges of the regulation mechanism of rice endosperm development, and provide some references for rice quality breeding.

水稻粉质胚乳突变体是解析胚乳发育及其调控机制的重要遗传材料。前期，我们分离了一个新的水稻粉质胚乳突变体ppr5，其胚乳蛋白含量降低，淀粉组分改变。PPR5定位于线粒体中，参与线粒体基因转录后加工，但PPR5突变引起粉质胚乳的发育机制尚不清楚。本项目拟以突变体和野生型发育胚乳为材料，开展：(1)利用细胞学形态技术查明PPR5突变对胚乳营养运输组织、胚乳组织的构建和分化、胚乳超微结构(重点是线粒体)等发育的影响；(2)利用生理生化和分子生物学方法，检测胚乳线粒体基因的转录产物和内含子的剪接效率，分析线粒体呼吸链复合物的丰度和活性及ATP含量的变化，阐明PPR5参与线粒体结构和功能的机制；(3)借助转录组学和分子生物学手段，揭示PPR5参与胚乳淀粉和蛋白合成、发育或积累的代谢通路。研究结果将阐明水稻PPR5蛋白突变引起粉质胚乳的发育机制，拓展对水稻胚乳发育调控机制的认知，为水稻品质育种提供参考。

胚乳作为水稻淀粉和蛋白质的储存器官，在很大程度上决定着稻米的产量和品质。水稻基因组中含有数百个PPR类蛋白，已被克隆的编码基因多是与叶色和育性相关，关于PPR蛋白调控水稻胚乳发育的作用机制仍尚不清楚。在该项目中，我们鉴定了一个新的水稻粉质不透明胚乳突变体ppr5。该突变体淀粉、蛋白和直链淀粉含量明显下降，支链淀粉精细结构和淀粉糊化特性也发生显著变化。半薄切片结果显示突变体发育胚乳中淀粉颗粒变小，复粒淀粉减少，多呈现单粒淀粉，糊粉层结构也发育异常。图位克隆和转基因功能互补结果证明PPR5基因编码一种P型PPR蛋白，含有七个PPR基序。PPR5在水稻各个组织中都有所表达，呈现组成型表达模式，在发育胚乳中随着籽粒灌浆表达量逐步降低，其编码蛋白定位于线粒体中。PPR5基因突变导致线粒体NADH脱氢酶4编码基因(nad4)内含子3的剪接效率降低，最终导致nad4的前体增加和成熟转录本缺失。nad4的剪接缺陷影响所在呼吸链复合体I的组装，导致表达丰度和活性下降，线粒体超微结构异常，ATP的合成受阻。此外，PPR5功能丧失后诱导交替氧化酶(AOXs)基因表达和编码蛋白含量显著上调。综上所述，该项目鉴定了一个新的P型PPR5蛋白，通过控制线粒体nad4内含子3的顺式剪接，参与线粒体功能和水稻胚乳发育。上述结果丰富和充实PPR蛋白家族的功能，拓展我们对水稻胚乳发育调控机制的认知，也为水稻品质育种提供理论依据。