水稻花器官数目异常突变体afon1的表型分析与基因定位

【目的】研究水稻花器官数目异常突变体afon1(abnormal floral organ number1)的分子机理,鉴定出控制水稻花器官数目变化的基因。【方法】利用甲基磺酸乙酯(EMS)诱变籼稻品种浙农34获得一个花器官数目异常突变体作为试验材料,命名为afon1。开花期随机取突变体afon1和野生型浙农34的稻穗各5个,利用组织学和扫描电子显微镜等技术研究afon1的花器官表型、细胞学特征和花粉育性。成熟期随机取突变体afon1和野生型浙农34植株各10株,测定株高、分蘖数、穗长、每穗颖花数、每穗实粒数和千粒重等农艺性状。随机取突变体afon1和野生型饱满种子各100粒,测定发芽势和发芽率。以突变体afon1为母本,分别与野生型浙农34和粳稻品种浙农大104杂交构建2个F2群体进行遗传分析和基因定位,筛选候选基因进行DNA测序比对,构建AFON1蛋白质的空间模型并对其结构进行分析,同时对候选基因以及与花器官数目相关的基因进行实时荧光定量PCR分析。【结果】与野生型相比,突变体afon1中59.64%小穗的花器官数目发生异常,其中多数小穗仅在内稃一侧产生一个颖壳状的器官,部分小穗表现2—4轮花器官数目同时增加;株高和千粒重显著增加,而结实率显著降低。遗传分析表明,突变体afon1与野生型浙农34杂交的F1植株小穗花器官数目表现正常,F2群体中小穗花器官数目正常植株与花器官数目异常植株的分离比符合3﹕1,表明突变体afon1性状受一对隐性核基因控制,基因位于水稻第1染色体长臂端In Del标记1M5和1M18之间,物理距离为73 kb,该区间内共有6个注释基因。突变体afon1和野生型的测序比对发现,突变体afon1中的基因LOC_Os01g67430外显子中第565个碱基T突变成A,导致第189个氨基酸由色氨酸突变为精氨酸。蛋白质序列和空间结构分析表明,AFON1蛋白质序列中含有一个Lipase_3结构域,结构域内的突变导致蛋白质的空间结构发生了明显的变化。实时荧光定量PCR结果显示,LOC_Os01g67430在突变体afon1幼穗中的表达量要显著高于野生型,而在根、茎和叶中则无显著差异;穗发育早期FON1和FON2/4等调控花器官数目的基因在突变体afon1花器官中的表达量显著增加。【结论】LOC_Os01g67430为突变基因afon1,该基因通过影响花器官数目相关基因的表达而调控各轮花器官数目。

Phenotypical Analysis and Gene Mapping of Abnormal Floral Organ Number Mutant afon1 in Rice (Oryza sativa L.)

【Objective】The abnormal floral organ number mutant of rice was used to study the molecular mechanisms of floral organ development, and to identify the related genes of floral organ in rice. 【Method】In present study, a rice mutant, abnormal floral organ number1 (afon1) was isolated from an indica cultivar Zhenong 34 M2 population by mutagenesis with ethyl methane sulfonate (EMS). At flowering stage, five panicles from afon1 and Zhenong 34 were randomly selected to observe the morphological phenotype, cytological features and pollen fertility by histology analysis and scanning electron microscopy, respectively. At mature stage, ten plants from afon1 and Zhenong 34 were randomly chosen for measuring the main agronomic traits, such as plant height, number of tillering, panicle length, number of floret per panicle, number of filled grain per panicle and 1 000-grain weight. One hundred plump seeds were selected for calculating the germination potential and germination rate of the mutant afon1 and wild type. The F2 population from crossing of afon1 with WT Zhenong 34 and Zhenongda 104 were used for genetic analysis and gene mapping, respectively. Then, the DNA sequencing was conducted, the model of AFON1 protein was built and the space structure for AFON1 protein was analyzed. Moreover, the expression of candidate gene and floral organ number associated genes were detected by real-time PCR. 【Result】Compared with the wild type, the floral organ number of 59.64% spikelet was abnormal in mutant afon1, which most of them had a glume-like organ on the side of the leman and there were 2-4 rounds of floral organ number increased at the same time in others. Furthermore, the plant height and 1 000-grain weight of afon1 were visibly higher, while the seed setting rate was significantly reduced. The results of the genetic analysis showed that the phenotype of F1 population from the crossing of afon1 with Zhenong 34 was normal and the segregation ratio of wild-type and mutant phenotype plants from F2 population fitted a ratio of 3﹕1, which revealed that the mutant trait of afon1 was controlled by a single recessive nuclear gene. The afon1 was further mapped on the arm of chromosome 1 between InDel markers 1M5 and 1M18 with a physical distance of 73 kb, where there were 6 annotated genes. The sequencing results between the mutant afon1 and the wild type illustrated that there was a single base-pair substitution of T (565th) to A on the exon of LOC_Os01g67430 resulting in the mutation of Trp (189th) to Arg. Protein sequencing and structure analysis revealed that there was a Lipase_3 domain and the mutation in the region changed the space structure of AFON1 obviously. The real-time PCR result showed that the expression of LOC_Os01g67430 in young panicle increased significantly, while the expression had no obvious difference in root, stem and leaf. Additionally, the expression of floral organ number related genes FON1 and FON2/4 was obviously increased in floral organ at young panicle developmental stage. 【Conclusion】The LOC_Os01g67430 was speculated as the gene afon1 regulating the number of floral organ by influencing the expression of corresponding genes which determined the number of floral organ.