水稻内颖畸形或缺失基因OsAPP1的图位克隆及表达分析

Map-based Cloning and Expression Analysis of Rice Abnormal or Absent Palea Gene OsAPP1

在育种基地材料中发现一株内颖畸形或缺失(abnormal or absent palea)突变体,将其命名为app1。该突变体在营养生长时期发育正常,但抽穗后突变体表现出内颖畸形(比外稃短导致颖壳不闭合,或者出现两个内稃)或缺失,其花粉育性为55.52%,结实率为6.48%,千粒重为10.811 g,种子发芽率为55.21%。以突变体app1与日本晴杂交构建了F1和F2群体,F1颖壳表型正常,F2群体出现内颖畸形和正常表型分离,内颖正常和突变表型分离比例为3∶1,表明app1内颖突变表型由单隐性核基因控制。以F2为分离群体,将app1精细定位于第3染色体上,位于分子标记ID4231和ID4246之间,遗传距离1.3 cM,对应物理距离为13.2 kb。该区段内完全包含1个开放阅读框,包含两个部分开放阅读框,经过测序分析发现候选基因LOC_Os03g11614启动子区发生点突变和245 bp缺失,qRT-PCR分析证实LOC_Os03g11614为OsAPP1基因。已有报道LOC_Os03g11614编码OsMADS1,是调控水稻花器官发育的重要明星基因,其不同位置的突变可以导致叶状颖壳和不育、以及控制籽粒大小。与3000份水稻种子资源SNP/Indel变异类型对比分析发现,突变体app1启动子的突变完全不同于现已OsMADS1研究报道突变类型,且与数据库中的自然突变类型多数不同。因此,本研究发现的app1突变体,是以往报道中从未出现的OsMADS1启动子发生突变的新型突变,且该类突变导致了其降低表达量,并产生了不同于前人研究的新表型,这为深入研究OsMADS1基因在水稻花器官发育中的功能提供了新的种质资源和思路。

This study reported the identification of a naturally-occurring mutant app1 which shows abnormal(shorter than the lemma,resulting in the unclosed shell or two palea)or absent palea at the reproductive stage.If compared with wild type,this mutant showed 55.52%of the pollen fertility,6.48%of the seed setting rate,10.811 gram of the thousand-grain weight as well as 55.21%of the seed germination rate.By generating the F1 and F2 populations crossed with Nipponbare,F1 plants was observed with a wild-type phenotype and F2 individuals were found with a 3∶1 segregation,thus indicating that the app1 mutant phenotype is controlled by a single recessive nuclear gene.Genetic mapping suggested that app1 was located on chromosome 3,flanked by molecular markers ID4231 and ID4246,with a genetic distance of 1.3 cM which corresponds to a physical distance of 13.2 kb.The physical interval contained a single complete open reading frame gene LOC_Os03 g11614 in which a point mutation and a 245 bp deletion were detected in the promoter region.RT-qPCR analysis revealed a reduction on the transcriptional level in app1 mutant,thus implying that LOC_Os03 g11614 is the most strong candidate.LOC_Os03 g11614 was known to encode for OsMADS1 that regulates the development of rice floral organs.Mutations in positions led to various phenotypic variations such as leafy glumes,sterility,and smaller grain size.By taking use of 3000 rice seed resources SNP/InDel mutation database,the mutation in the app1 promoter of OsMADS1 is unique and a novel allele,which particularly resulted in a reduction on gene expression.Thus,this study provided a new germplasm resource which is useful to decipher the OsMADS1 function in the formation of rice floral organs.