水稻507ys黄绿叶突变体的遗传鉴定与候选基因分析

Genetic Identification and Candidate Gene Analysis of Yellow-Green Leaf Mutant 507ys in Rice

【目的】对水稻507ys黄绿叶突变体进行遗传鉴定与候选基因分析。【方法】用化学诱变剂甲基磺酸乙酯(EMS)处理粳稻品种日本晴(Nipponbare),从突变体库中获得一份黄绿叶突变体507ys。对该突变体进行表型观察以及主要农艺性状调查分析。将507ys与正常绿色品种进行杂交,调查F1代的叶色表型和F2群体的叶色分离情况,分析该突变表型的遗传行为。利用(507ys/明恢63)的F2作为定位群体,对507ys突变基因进行精细定位且遴选候选基因,对候选基因进行DNA测序验证及编码蛋白序列同源性分析。同时,测定507ys突变体和野生型亲本的光合色素含量,并利用高效液相色谱(HPLC)精确分析它们的叶绿素组成成分,以进一步揭示507ys黄绿叶突变基因的候选基因。【结果】507ys黄绿叶突变体整个生育期呈黄绿色。与野生型亲本日本晴相比,507ys突变体在分蘖期叶片的叶绿素和类胡萝卜素含量分别下降52.1%和58.1%,成熟期株高、每株有效穗数、每穗着粒数和结实率分别减少8.3%、51.0%、7.4%和11.6%。507ys与正常绿色品种日本晴和明恢63杂交的F1表现正常的绿色,F2群体绿色正常植株与黄绿叶突变植株分离比符合3﹕1,表明507ys的黄绿叶突变性状由1对隐性核基因控制。该突变基因定位在第10染色体长臂近端部SSR标记RM333和InDel标记L3之间,遗传距离分别为0.56 cM和0.14 cM,两标记之间的物理距离约为60.2 kb,此区间内包含13个有注释的预测基因。基因组序列分析发现,507ys突变体中编码叶绿素酸酯a加氧酶的OsCAO1(LOC_Os10g41780)在编码区第2 198位碱基(CDS第1 057位碱基)处,碱基G突变为碱基A,造成编码蛋白的氨基酸序列第353位的谷氨酸(E)突变成赖氨酸(K)。对叶绿素组成成分分析表明,507ys突变体叶片中只有叶绿素a,没有叶绿素b。【结论】507ys突变体基因是已报道的叶绿素酸酯a加氧酶基因OsCAO1的等位基因。507ys突变体在OsCAO1外显子上发生的一个点突变使得其体内叶绿素酸酯a加氧酶失活,造成叶绿素b合成受阻。

Abstract： [Objective] The present study was conducted aiming at genetic identification and candidate gene analysis of yellow-green leaf mutant 507ys. [Method] A yellow-green leaf mutant, designated as 507ys, was isolated from the progeny of a japonica rice cv. Nipponbare treated with ethyl methanesulfonate. Phenotypes of the 507ys mutant were observed and its main agronomic traits were analyzed under field conditions inChengdu, Sichuan. After the 507ys mutant was crossed with normal green varieties, leaf colour phenotypes of the F1 progenies and the segregation ratio of yellow-green and green leaf plants in the F2populations were investigated. Genetic mapping of the mutant gene was conducted using 360 yellow-green leaf individuals from the F2 mapping population of 507ys/Minghui63. Putative genes in the fine mapped region were analyzed, and the candidate genes in the mutant and its wild-type were sequenced, respectively. Alignment of the deduced amino acid sequences of homologous OsCAO1 proteins was conducted. In addition, contents of photosynthetic pigments in the 507ys mutant and its wild-type were determined by spectrophotometer, and their chlorophyll compositions were well-examined by high-performance liquid chromatography （HPLC） so that the candidate gene resulting in the 507ys mutant phenotype could be further identified. [Result] The whole plant of the mutant exhibited yellow-green leaf trait throughout the growing period. Compared with its wild-type parent Nipponbare, the contents of chlorophyll and carotenoid decreased by 52.1% and 58.1%, and plant height, the number of productive panicles per plant, the number of spikelets per panicle and seed setting rate reduced by 8.3%, 51.0%, 7.4% and 11.6%, respectively. All FI plants generated by crossing yellow-green leaf mutant 507ys with normal green varieties Nipponbare and Minghui 63 showed normal green leaf. The segregation ratios of normal leaf plants and yellow-green leaf plants in two F2 populations both 3：1, indicating the 507ys mutant phenotype is controlled by a single recessive nuclear gene. The mutant gene was finally mapped to a region of 60.2 kb between SSR marker RM333 and InDel marker L3 on the long arm near the end of chromosome 10, in which thirteen predicted genes were annotated in the Rice Genome Annotation Project. Sequencing analysis of these candidate genes between the mutant and its wild-type revealed the single base change （G2198A） in the coding region of the OsCA01 （LOCOs10g41780） gene for chlorophyllide a oxygenase resulted in a missense mutation （E353K） in the encoded product. HPLC analysis of chlorophyll composition indicated the 507ys mutant accumulated only chlorophyll a and no chlorophyll b. [ Conclusion] The 507ys mutant gene was allelic to OsCA01 gene. A point mutation in exons of OsCA01 gene in the 507ys mutant makes chlorophyllide a oxygenase inactive, resulting in the block in chlorophyll b biosynthesis.