鸟苷酸激酶OsGK1对水稻种子发育至关重要

The Guanylate Kinase OsGK1 is Essential for Seed Development in Rice

【目的】对水稻粉质皱缩突变体fse2进行表型分析及基因克隆,为阐明水稻淀粉合成机制以及胚的发育奠定基础。【方法】fse2来自粳稻品种滇粳优1号的MNU(N-甲基-N-亚硝基脲)诱变突变体库。本研究考查了突变体fse2籽粒的理化性状,利用扫描电镜和半薄切片观察了淀粉颗粒的结构;构建了fse2与N22的F_2群体,通过图位克隆及转基因互补验证确定目标基因;通过qRT-PCR以及GUS活性染色对FSE2进行组织表达分析;免疫印迹分析了突变体中淀粉合成相关基因以及线粒体基因的蛋白变化。【结果】fse2籽粒粉质皱缩,千粒重显著下降;胚乳中淀粉颗粒变小变圆,排列松散,不能形成正常的复合淀粉颗粒;突变体中总淀粉、直链淀粉含量均显著下降,脂肪含量显著上升,突变体淀粉的糊化特性发生明显改变。FSE2编码一个线粒体和质体双定位的鸟苷酸激酶(guanylate kinase),命名为OsGK1。OsGK1在各器官中组成型表达,并在花后6 d的胚乳中表达水平最高。突变体胚乳中淀粉合成相关蛋白水平显著降低,尤其是AGPS2b和PHOI。此外,突变体fse2的胚发育严重受损,导致种子纯合致死;线粒体定位的AOX积累显著增强,而野生型中几乎检测不到,表明线粒体呼吸途径受损。【结论】由于OsGK1的功能缺陷,导致水稻种子中线粒体和造粉体发育异常,进而产生了胚致死以及胚乳粉质皱缩的表型,因此OsGK1对水稻种子的发育至关重要。

【Objective】In this study,the phenotype of the floury and shrunken endosperm mutant fse2 was analyzed.Isolation of the responsible gene will lay a foundation for elucidating the mechanism underlying starch synthesis and embryo development in rice.【Method】fse2 was obtained from the mutant library of japonica cultivar Dianjingyou 1 induced with N-Nitroso-N-methylurea.In this study,the physiochemical properties of fse2 endosperm were investigated and the structure of starch grains was observed.An F2 population derived from fse2 and N22 was constructed,then the underlying gene was determined by map-based cloning and complementation tests.qRT-PCR and GUS staining were used to analyze the expression of FSE2.Western blotting was performed to analyze the protein levels of starch synthesis related genes and mitochondrial genes in the mutant.【Result】Compared with the transparent endosperm of wild type,fse2 displayed a floury and shrunken endosperm,significantly declined 1000-grain weight,smaller and loosely packed irregular compound starch grains.Total starch and amylose content decreased significantly,while the lipid content increased obviously in fse2,and the gelatinization characteristics of fse2 were changed notably.FSE2 encodes a guanylate kinase named OsGK1,which is dual-targeted to both mitochondria and plastids.OsGK1 was constitutively expressed in various organs with the highest level in developing endosperm at 6th day after flowering.The protein levels of most of the starch synthesis related genes in the mutant endosperm were significantly decreased,especially AGPS2b and PHOI.In addition,the homozygous seeds of fse2 were lethal and the development of fse2 embryos was severely arrested.The accumulation of mitochondria AOX was notably elevated,while almost undetectable in the wild type,indicating the mitochondrial respiratory chain was impaired.【Conclusion】Due to the functional defects of OsGK1,the development of mitochondria and amyloplasts are abnormal,leading to the embryo lethality and a floury and shrunken endosperm.