水稻白条纹突变体6001的基因精细定位与候选基因筛选

发布时间：2018-06-02 08:43

本文选题：水稻 + 白条纹　；参考：《南京师范大学》2017年硕士论文

【摘要】：水稻叶片光合作用能力决定了生物产量和经济产量。研究水稻叶色突变体光合作用的相关机制对提高光合效率和培育高光效水稻品种提供理论基础,水稻叶色突变体性状明显,来源广泛,是功能基因组学研究的重要材料,对阐明叶绿素代谢合成途径、叶绿体发育的分子遗传机理具有重要意义。本研究对一份来源于籼稻品种9311的叶色突变体,进行了性状分析和图位克隆研究,研究结果表明:1.该叶色突变体从播种伊始表现为白化,三叶期后白化叶片逐渐转成白绿相间的条纹叶,从叶缘向中脉白条与绿条相间排列,叶鞘也呈白条纹状,低温条件下叶片白化现象更明显。整个水稻生长过程中,叶片白色部分相比绿色部分逐渐减少。抽穗期直至蜡熟前期,颖壳也出现白化现象。2.选取突变体6001和野生型9311分蘖期和抽穗期叶片测定叶绿素含量。在分蘖期,突变体各色素叶绿素a、叶绿素b、胡萝卜素含量分别为正常材料的50%、65%、52%;在抽穗期,突变体叶绿素a、叶绿素b、胡萝卜素色素含量分别为正常材料的54%、58%、62%。3.用粳稻02428与突变体6001杂交F2代200个隐性单株进行突变基因定位,初步将基因定位于第6染色体SSR标记RM136与RM19715之间,再进一步用白条纹突变体6001/02428杂交F2的1300株隐性单株精细定位,在水稻第6染色体设计微卫星(SSR)以及Indel标记,通过构建的近等基因池进行多态性标记的筛选,共筛选出12对多态性标记,对这些标记在F2代隐性单株中的带型进行统计发现,距离目标基因最近的2个标记分别是Y27和RM19732,并分别检测出9个和1个重组个体。2分子标记之间的物理距离为75 kb,并与标记Y26共分离。4.利用测序和生物信息学分析,发现该区域内共有12个注释基因。对可能与突变体性状相关的候选基因测序比对,发现基因LOC_Os06g14620 (核糖核苷酸还原酶小亚基)的编码区包括第1个起始密码子碱基在内共有460bp碱基缺失,且在起始密码子上游也缺失134bp碱基,推测是st1的等位基因。基因的编码区虽有460bp缺失,但保留部分仍存在起始密码子ATG和终止密码子TGA,可能只翻译出有部分功能的蛋白。
[Abstract]:The photosynthesis ability of rice leaves determines the biological yield and economic yield. Studies on the mechanism of photosynthesis of rice leaf color mutants provide theoretical basis for improving photosynthetic efficiency and cultivating rice varieties with high photosynthetic efficiency. Leaf color mutants of rice have obvious characters and wide sources, which are important materials for functional genomics research. It is important to clarify the pathway of chlorophyll metabolism and the molecular genetic mechanism of chloroplast development. In this study, a leaf color mutant from indica rice variety 9311 was studied by character analysis and map cloning. The results showed that: 1. The leaf color mutant showed albinism from the beginning of seeding. After the trefoil stage, the albino leaf was gradually transformed into striped leaves with white and green phase, and the leaf sheath was also white stripe, which was arranged from leaf margin to midvein white stripe and green stripe. The phenomenon of leaf whitening was more obvious at low temperature. During the whole rice growing process, the white part of the leaves gradually decreased compared with the green part. At heading stage to the early stage of wax ripening, the glume also appeared albinism. 2. Chlorophyll content in leaves of mutant 6001 and wild type 9311 were determined at tillering stage and heading stage. At tillering stage, the contents of chlorophyll a, chlorophyll b and carotene of the mutants were 50%, 65% and 52% of normal materials, respectively, and at heading stage, the contents of chlorophyll a, chlorophyll b and carotene pigments of the mutants were 54 / 622 路3, respectively, of the normal materials. Two hundred recessive single plants of F _ 2 generation of japonica rice 02428 and mutant 6001 were used to map the mutant genes. The gene was preliminarily mapped between RM136 and RM19715 on chromosome 6, and 1300 recessive plants of F _ 2 crossed with white stripe mutant 6001 / 02428 were further mapped. Microsatellite SSRs and Indel markers were designed on chromosome 6 of rice. Twelve pairs of polymorphic markers were screened by screening the constructed near isogenic pool, and the band patterns of these markers in F2 recessive single plant were statistically found. The two nearest markers to the target gene were Y27 and RM19732, respectively. The physical distance between the two markers was 75 kband and separated from the marker Y26. A total of 12 annotated genes were found in the region by sequencing and bioinformatics analysis. By sequencing the candidate genes that may be related to mutant traits, we found that the coding region of LOC_Os06g14620 (ribonucleotide reductase small subunit) shared 460bp base deletion, including the first start codon base. The 134bp base was also absent in the upstream of the start codon, which is presumed to be the allele of st1. Although there is 460bp deletion in the coding region of the gene, there is still a start codon ATG and a termination codon in the reserved part, which may only translate some functional proteins.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511

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