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小麦大粒突变体的遗传分析和相关基因片段的克隆

Genetics Analysis and Cloning of Associated Gene Fragments for Large Grain Size Mutant in Wheat

作者: 专业:遗传学 导师:李斯深 年度:2009 学位:硕士  院校: 山东农业大学

Keywords

Wheat, EMS, Mutant, EST-SSR, SSR, SSH

        传统的化学诱变方法,能诱发产生高密度的点突变,获得遗传背景相似的突变体。不仅能够解决小麦育种中种质资源匮乏的问题,也为相关基因的精细定位、克隆以及基因功能的分析等提供了基础材料。本研究采用EMS诱变小麦品种烟农15获得的大粒、高杆突变体8008为研究材料,对发生突变的粒长、粒宽和千粒重等性状进行遗传分析,尝试对突变基因进行初步定位及基因片段的克隆,为从分子水平上揭示小麦粒重遗传机制奠定基础。(1)大粒、高秆突变体8008与受体烟农15杂交,构建遗传分析群体。通过对杂种F2代和F3代群体的表型分析发现,高千粒重与低千粒重、宽粒与窄粒、高杆和矮杆这三对性状在F2群体中均呈3:1的分离比例,并且经卡方检验差异极显著,均为显性单基因控制;相关性分析表明,粒宽与粒重、粒长与粒重、高杆与粒重之间均为极显著相关。(2)用突变体8008和烟农15对860对EST-SSR引物和960对SSR引物进行筛选,所得差异引物在F2群体中选株进行二次筛选,共获得59对引物在群体中能扩增出99条差异条带。这59对引物共分布在16条染色体上,以B染色体组上分布位点最多,共27个;在16条染色体中,1B、2B、7B三条染色体上分布的位点最多,分别有7个、6个、6个。(3)以突变体8008和受体烟农15分别做为试验方和驱动方,利用抑制消减杂交技术(SSH),构建双向差减文库。在双向库中随机挑取阳性克隆进行测序,并在GenBank数据库中进行生物信息学分析。共获得12条EST与已知功能基因高度同源,主要包括:与光合作用相关的基因如RuBP羧化酶、光敏色素、叶绿素等;与物质代谢作用相关的基因如半胱氨酸水解酶、半胱氨酸蛋白酶、果糖基转移酶、蔗糖合酶I等;与RNA功能相关的基因如RNA结合蛋白、核糖蛋白体等。5条EST序列与未知功能的禾本科作物cDNA片段高度同源,7条EST未能找到同源性匹配。
    The traditional method of chemical mutagenesis can produce high-density induced mutations with similar genetic background. This can not only solve the problem of poor germplasm in wheat breeding, but also provides a research platform for the fine positioning, cloning, and functional analysis of related genes. In this study, we have got the mutant called large grain size and long-culm line 8008 by treated the wheat variety“Yannong 15”with EMS (ethyl methane sulfonate). Taking the mutant line 8008 as experiment material, we went on the genetic analysis of mutant character including grain longth, grain width and grain weight, tried to map these mutant gene and clone associated gene fragment, and this could lay a foundation for forther revealing the genetic mechanism of grain weight heredity in wheat.(1) Cross the large grain size and long-culm line 8008 with Yannong15 to construct the population for genetic analysis. Through the phenotype analysis of F2 and F3 population, we found that the traits high grain weight & low grain weight, wide grain & narrow grain, long-culm & short-culm showed a 3:1 ratio of the separation, and differences by chi-square test was significant, which shows that all these three pairs of traits are controlled by single dominant gene. Correlation analysis showed that grain width and grain weight, grain length and weight, long-culm and weight are among the most significant correlation(2) We look for polymorphic markers from 860 EST-SSR and 960 SSR primer pairs by amplying in line 8008 and Yannong 15. These polymorphic markers were amplied in F2 population for forther analysis. Finally, a total of 99 polymorphic fragments were detected from 59 primer pairs, which were mainly distributed over 16 chromosomes. Most of these markers (27 primer pairs) were mapped on genome B; and there were 7, 7 and 6 primer pairs which were mapped on chromosome 1B, 2B and 7B, respectively.(3) The mutant line 8008 and Yannong 15 are used as tester and driver respectively for the structure of forward and reverse suppression subtractive hybridization cDNA libraries. Positive clones are randomly chosen from each of the two libraries and then sequenced, and propose sequence alignment in GenBank by BlastN in GenBank. A total of 12 ESTs which was highly homologous with the known functional genes were obtained. These functional genes could be grouped into 3 parts., include genes associated with photosynthesis, such as RuBP carboxylase, phytochrome, chloroplast, etc.; and material metabolism-related genes, such as homocysteine hydrolase, cysteine protease, fructosyltransferase, sucrose synthase type I, etc.; and genes of RNA function such as RNA-binding protein, ribosomal protein, and so on. There are 5 EST sequences which are highly homologous with unknown function of Gramineae crops cDNA fragments,and seven ESTs fail to find homology EST match.
        

小麦大粒突变体的遗传分析和相关基因片段的克隆

缩略词3-7
摘要7-8
ABSTRACT8-9
1 引言10-26
    1.1 人工诱变产生突变体11-14
        1.1.1 EMS 诱变机制11
        1.1.2 EMS 诱变的特点11-12
        1.1.3 EMS 诱变的应用12-13
        1.1.4 突变体库的构建与应用13-14
    1.2 图位克隆14-19
        1.2.1 图位克隆的技术环节15-17
        1.2.2 图位克隆的应用17-18
        1.2.3 图位克隆应用的局限性18-19
    1.3 抑制消减杂交技术19-25
        1.3.1 抑制消减杂交技术(SSH)原理和特点19-20
        1.3.2 抑制性差减杂交(SSH)技术的基本步骤20-22
        1.3.3 抑制消减杂交(SSH)在植物中的应用22-25
    1.4 本研究意义、研究内容、预期目标25-26
2 材料与方法26-43
    2.1 实验材料26-27
        2.1.1 植物材料26
        2.1.2 主要试剂26
        2.1.3 PCR 引物26-27
    2.2 实验方法27-31
        2.2.1 农艺性状调查27
        2.2.2 突变体的遗传分析27
        2.2.3 突变体的分子标记分析27-31
    2.3 抑制消减杂交(SSH)31-40
        2.3.1 植物材料31
        2.3.2 SSH 用试剂耗材31-32
        2.3.3 总RNA 抽提32-33
        2.3.4 RNA 纯化及质量检测33
        2.3.5 mRNA 分离纯化33-34
        2.3.6 cDNA 反转录34-35
        2.3.7 cDNA RsaI 酶切35-36
        2.3.8 接头连接36-37
        2.3.9 差减杂交37-38
        2.3.10 二轮差减PCR 鉴定38-40
    2.4 差异片段的克隆40-43
        2.4.1 差减PCR 扩增产物纯化40
        2.4.2 差异片段的连接40-41
        2.4.3 转化41-42
        2.4.4 测序及同源性比较42-43
3 结果与分析43-57
    3.1 突变性状的遗传分析43-45
    3.2 分子标记分析45-47
    3.3 大粒突变体的SSH 分析47-57
        3.3.1 总RNA 的提取和纯化47-48
        3.3.2 逆转录及Rsa I 酶切结果检测48-49
        3.3.3 二次PCR 后的消减杂交产物检测49
        3.3.4 差异表达基因的克隆与EST 序列分析49-57
4. 讨论57-61
    4.1 EMS 诱变获得大粒突变体57-58
    4.2 粒重相关基因的定位58-59
    4.3 SSH 的应用59-61
结论61-62
参考文献62-72
致谢72-73
攻读学位期间发论文表情况73
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