水稻胚乳突变体b140的表型分析及基因克隆

Phenotyping and gene cloning of a floury endosperm mutant b140 in rice

[目的]对水稻粉质胚乳突变体b140进行表型分析和基因克隆,为阐明水稻淀粉合成机制奠定基础。[方法]以粳稻品种‘W017’为野生型,测量‘W017’和突变体b140的农艺性状,分析籽粒的灌浆期干物质积累量和理化性质,并观察淀粉颗粒的结构;构建b140与‘南京11’的F2群体,取隐性极端个体定位基因;利用qRT-PCR测定籽粒灌浆时期的淀粉合成相关基因表达量;测定相关淀粉合成酶活性。[结果]b140表现为粉质胚乳,粒宽、粒厚和千粒质量均显著低于野生型,但粒长和其他农艺性状无显著变化。b140种子的总淀粉和直链淀粉含量均显著低于野生型,而可溶性糖含量显著高于野生型。扫描电镜观察b140胚乳淀粉粒呈球形或不规则形状,排列疏松。突变基因定位在第1号染色体短臂56 kb的区间内,测序发现只有1个已知基因Os01g0179400发生突变。qRT-PCR结果显示,与野生型相比,b140胚乳中淀粉合成相关基因的表达量出现不同程度的下调。b140胚乳中腺苷磷酸葡萄糖焦磷酸化酶的活性显著低于野生型。[结论]b140由于Os01g0179400基因发生突变,导致粉质胚乳表型。Os01g0179400突变影响了淀粉合成相关基因的表达以及腺苷磷酸葡萄糖焦磷酸化酶的活性。b140的发现对于了解水稻淀粉合成和调控的机制具有一定的意义。

[ Objectives ] We analyzed the phenotype of a rice floury endosperm mutant b140 and cloned this gene to lay the foundation for the mechanism of starch biosynthesis. [ Methods] Related agronomic traits, dry matter accumulation during grain filling and physi- cochemical properties of wild type and b140 grains were measured and the structure of starch granules were observed. A hybrid F2 population of b140 and＇ Nanjing 11 ＇ was developed and the recessive individuals were selected to map the locus, qRT-PCR was conducted to analyze the expression of genes related to starch biosynthesis. The activities of related enzymes were measured. [ Results ] The grain width, grain thickness and 1 000-grain weight of b140 significantly decreased compared with the wild type. There was no detectable change in grain length and other agronomic traits of b140 compared with the wild type. Total starch content and amylose content in mature seeds of b140 decreased compared with wild-type. The content of soluble sugars was significantly higher than that of wild type. Scanning electron microscopy analysis showed that the mutant endosperm contained loosely packed irregular compound starch granules. The b140 locus was delimited to a 56 kb region on Chromosome 1. Sequence analysis revealed that the coding region of OsOlgO179400, an already reported gene, had a mutation. The results of qRT-PCR indicated that the expression levels of some genes participating in starch synthesis were down-regulated in b140. The analysis of enzymatic activity showed that the activity of adenosine diphosphoglucose pyrophosphprylase in b140 was significantly lower than that in wild type. [ Conclusions ] OsO1gO179400 contained a single nucleotide substitution in bl40,leading to floury endosperm. OsOlgO179400 might play an impor- tant role in the synthesis and regulation of starch in rice and affect the activity of adenosine diphosphoglueose pyrophosphprylase. The discovery of b140 has certain significance for the understanding of starch synthesis and regulation mechanism.