水稻粉质胚乳基因的精细定位与功能分析

发布时间：2018-03-31 02:05

本文选题：水稻　切入点：胚乳　出处：《西北农林科技大学》2017年硕士论文

【摘要】：本研究从烷化剂N-甲基N-亚硝基脲(MNU)处理的韩国粳稻品种Hwacheong突变体库中,经筛选得到一个稳定遗传粉质胚乳突变体flo(t)。首先对该突变体进行基本表型鉴定及理化性质分析,然后对淀粉的理化性质和结构特性进行分析,并利用图位克隆手段对目标基因进行精细定位、基因克隆和功能验证分析。主要实验结果如下:1.flo(t)突变体籽粒的胚乳表现为白色不透明粉质状,突变体种子粒长没有明显变化,粒宽和粒厚有所降低,千粒重明显降低。胚乳横截面扫描电镜观察显示flo(t)淀粉粒之间距离变大,排列疏松,而野生型排列紧密充实。但是两者的淀粉粒形状差异不大。生理生化测定结果显示:突变体种子的总淀粉含量和直链淀粉含量较野生型均降低,尤其是总淀粉含量,明显下降(17%)。蛋白质含量、脂质含量较野生型相比均有提高。2.对突变体淀粉特性研究表明:突变体分子结构发生变化,支链淀粉中连接多个簇的长B链含量显著降低;结晶度降低,野生型结晶度为29.96,而突变体结晶度下降到23.63;热力学特性分析显示突变体的糊化起始温度降低;突变体的链长分布发生变化,其聚合度为8-14的中短链分支比例增加,DP3-7的短链、DP16-20的中短链和DP25-37的中长链减少。3.利用突变体与中花11杂交获得F1杂交种,F1自交获得F2群体进行遗传分析,显示后代表型分离比为3:1。表明flo(t)突变表型是由单隐性基因控制。利用突变体与籼稻品种Dular获得F1杂交种,F1自交获得F2群体作为基因的定位群体。利用SSR、Indel和dCAPS标记将基因定位于第6号染色体上94.5kb的基因组区间。4.经水稻基因组数据库(Rice Genome Annotation Project)查询发现该区间有16个预测的开放阅读框(ORF),通过基因组序列测序结果发现ORF14(LOC_Os06g13810)在内含子剪接区域发生点突变,即由G变为A,RT-PCR结果显示该基因的cDNA序列有7个碱基的插入,导致该基因序列编码蛋白提前终止。5.构建含有该基因全长CDS的转基因互补载体,以flo(t)突变体作为受体进行水稻基因转化,结果表明转基因植株的种子都恢复透明表型,扫描电镜观察也证明了其淀粉粒由无序状态转变为排列较为紧密的状态。利用激光共聚焦显微镜观察瞬时表达该基因的本氏烟叶片发现,该基因编码的蛋白定位于细胞质中。
[Abstract]:In this study, a stable powdery endosperm mutant, flottl, was obtained from the Hwacheong mutants of Korean japonica rice treated with N-methyl-N-nitroso (MNU-N). The basic phenotype and physicochemical properties of the mutant were identified and analyzed. Then, the physicochemical and structural properties of starch were analyzed, and the target gene was mapped by map cloning. Gene cloning and functional analysis. The main results were as follows: 1. The endosperm of the mutant showed white opacity, the seed length of the mutant did not change obviously, and the grain width and grain thickness decreased. The scanning electron microscope observation of endosperm cross section showed that the distance between starch grains increased and the arrangement was loose. The results of physiological and biochemical tests showed that the total starch content and amylose content of the mutant seed were lower than that of the wild type, especially the total starch content. The protein content and lipid content were all increased compared with the wild type. The results showed that the molecular structure of the mutant changed and the long B chain content of multiple clusters in amylopectin decreased significantly. The crystallinity of wild type was 29.96, while the crystallinity of mutant was 23.63.The thermodynamic analysis showed that the initial temperature of gelatinization of mutant decreased, and the distribution of chain length of mutant changed. The proportion of short chain branching with degree of polymerization 8-14 increased, and that of short chain DP16-20 and DP25-37 decreased .3.The F _ 1 population of F _ 1 hybrid was obtained by crossing the mutant with Zhonghua 11, and the F _ 2 population was obtained by self-crossing of F _ 1 hybrids, and the genetic analysis was carried out in F _ 2 population. The segregation ratio was 3: 1, which indicated that the phenotype of flot1 mutation was controlled by single recessive gene. F _ 1 hybrid F _ 1 self-bred with indica rice variety Dular was used to obtain F _ 2 population as the localizing population. SSR-Indel and dCAPS markers were used to localize the gene. The gene was mapped to the genome interval of 94.5kb on chromosome 6. The rice genome database, Rice Genome Annotation Project, found that there were 16 predicted open reading frames in this region, and ORF14LOCOs06g13810 was found in the region by sequencing the genome sequence. A point mutation occurs in the splicing region. The results of RT-PCR showed that the cDNA sequence of the gene was inserted with 7 bases, which led to the early termination of the gene coding protein. The transgenic rice gene was transformed with flot mutants as the receptor. The results showed that the seeds of the transgenic plants all returned to transparent phenotype. Scanning electron microscopy (SEM) also showed that the starch grains changed from disordered state to more closely arranged state. Using confocal laser microscope to observe the transient expression of the gene in tobacco slices, it was found that the protein encoded by the gene was located in the cytoplasm.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511

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