本文選題：水稻　切入點：胚乳　出處：《西北農林科技大學》2017年碩士論文

【摘要】：本研究從烷化劑N-甲基N-亞硝基脲(MNU)處理的韓國粳稻品種Hwacheong突變體庫中,經篩選得到一個穩(wěn)定遺傳粉質胚乳突變體flo(t)。首先對該突變體進行基本表型鑒定及理化性質分析,然后對淀粉的理化性質和結構特性進行分析,并利用圖位克隆手段對目標基因進行精細定位、基因克隆和功能驗證分析。主要實驗結果如下:1.flo(t)突變體籽粒的胚乳表現為白色不透明粉質狀,突變體種子粒長沒有明顯變化,粒寬和粒厚有所降低,千粒重明顯降低。胚乳橫截面掃描電鏡觀察顯示flo(t)淀粉粒之間距離變大,排列疏松,而野生型排列緊密充實。但是兩者的淀粉粒形狀差異不大。生理生化測定結果顯示:突變體種子的總淀粉含量和直鏈淀粉含量較野生型均降低,尤其是總淀粉含量,明顯下降(17%)。蛋白質含量、脂質含量較野生型相比均有提高。2.對突變體淀粉特性研究表明:突變體分子結構發(fā)生變化,支鏈淀粉中連接多個簇的長B鏈含量顯著降低;結晶度降低,野生型結晶度為29.96,而突變體結晶度下降到23.63;熱力學特性分析顯示突變體的糊化起始溫度降低;突變體的鏈長分布發(fā)生變化,其聚合度為8-14的中短鏈分支比例增加,DP3-7的短鏈、DP16-20的中短鏈和DP25-37的中長鏈減少。3.利用突變體與中花11雜交獲得F1雜交種,F1自交獲得F2群體進行遺傳分析,顯示后代表型分離比為3:1。表明flo(t)突變表型是由單隱性基因控制。利用突變體與秈稻品種Dular獲得F1雜交種,F1自交獲得F2群體作為基因的定位群體。利用SSR、Indel和dCAPS標記將基因定位于第6號染色體上94.5kb的基因組區(qū)間。4.經水稻基因組數據庫(Rice Genome Annotation Project)查詢發(fā)現該區(qū)間有16個預測的開放閱讀框(ORF),通過基因組序列測序結果發(fā)現ORF14(LOC_Os06g13810)在內含子剪接區(qū)域發(fā)生點突變,即由G變?yōu)锳,RT-PCR結果顯示該基因的cDNA序列有7個堿基的插入,導致該基因序列編碼蛋白提前終止。5.構建含有該基因全長CDS的轉基因互補載體,以flo(t)突變體作為受體進行水稻基因轉化,結果表明轉基因植株的種子都恢復透明表型,掃描電鏡觀察也證明了其淀粉粒由無序狀態(tài)轉變?yōu)榕帕休^為緊密的狀態(tài)。利用激光共聚焦顯微鏡觀察瞬時表達該基因的本氏煙葉片發(fā)現,該基因編碼的蛋白定位于細胞質中。
[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.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S511

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