水稻粉质胚乳突变体cse的表型分析及基因定位

Phenotypic Analysis and Map-Based Cloning of Chalkiness and Floury Endosperm Mutant cse in Rice(Oryza sativa L.)

利用突变体挖掘与稻米品质相关基因,有助于阐明与品质相关的淀粉和贮藏蛋白生物合成的调控机制,促进水稻优质育种。通过筛选中花11组织培养突变体库,获得具有粉质、垩白和皱缩外观胚乳的突变体cse(chalkiness and shrunken endosperm),并进行籽粒表型鉴定和理化性质分析。通过F2群体分析cse遗传行为和目的基因的图位克隆,利用qRT-PCR分析基因的时空表达模式。与野生型相比,突变体的农艺性状和淀粉理化性质均有显著差异,突变体淀粉颗粒呈不规则球型,且相互间隙较大。遗传分析表明,突变体性状受单隐性核基因控制。借助图位克隆将候选基因定位在4号染色体长臂端Os4-14-8和Os4-15之间的40kb范围内。测序结果表明,定位区间内仅有一个编码TPR蛋白的候选基因LOC_Os04g55230发生了突变。第1个外显子处缺失3个碱基GGC,导致一个甘氨酸缺失,第14个外显子处存在1个碱基G替换成碱基A,导致精氨酸突变为赖氨酸。qRT-PCR结果表明,LOC_Os04g55230在授粉后14d的胚乳中表达量达到最大值。突变的LOC_Os04g55230为已报道基因OsFLO2/OsCNY8的新等位基因,但突变体与已知的OsFLO2突变体表型不完全相同,cse籽粒伴有皱缩外观,有效分蘖数、贮藏蛋白和脂肪酸含量等均显著增加。

It is helpful to clarify the genetic regulatory mechanism of starch and storage protein biosynthesis related to quality and promote rice quality breeding by using mutants related to rice quality.The chalkiness and shrunken endosperm mutant named cse was obtained by screening a mutant library of tissue culture of Zhonghua 11,and the phenotype identification and physicochemical properties analysis of grains were carried out.Analysis of genetic behavior of cse and map-based cloning of target gene by F2 population.qRT-PCR was used to analyze the spatiotemporal expression pattern of related genes.Compared with the wild type,the mutant showed significantly different in agronomic traits and starch physicochemical characteristics.The mutant starch granules were irregularly spherical with large interspaces.Genetic analysis showed that the mutant trait was controlled by a pair of recessive nuclear genes.Map-based cloning revealed that cse is located on the long arm of chromosome 4 between Os4-14-8 and Os4-15 with a physical interval of 40kb.Sequencing results showed that only one candidate gene LOC_Os04g55230 encoding TPR protein had mutation in the mapping interval.The deletion of three bases GGC in the first exon led to the deletion of one glycine,and the substitution of one base G in the fourteenth exon for base A led to the mutation of arginine to lysine.The results of qRT-PCR showed that the expression level of LOC_Os04g55230 reached the maximum in endosperm 14 days after fertilization.The mutant LOC_Os04g55230 was a new allele of the reported gene OsFLO2/OsCNY8,but the phenotype of the mutant was not completely same as the known OsFLO2 mutant.The cse grains had shrunken appearance,and the effective tiller number,storage protein and fatty acid content were significantly increased.