【作者】 赵永涛；

【导师】 詹克慧； 孔秀英；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 低分子量麦谷蛋白是小麦谷蛋白的主要组成部分(约占60%),在很大程度上决定着小麦的加工品质。编码LMW-GS的基因位于小麦第一同源群1A、1B和1D染色体短臂上,分别称为Glu-A3、Glu-B3和Glu-D3(统称Glu-3)。低分子量麦谷蛋白亚基受多基因控制,并且在染色体上与醇溶蛋白基因紧密连锁,在SDS-PAGE上又不易与醇溶蛋白区分,所以低分子量麦谷蛋白的研究存在着很大难度。本研究以四倍体小麦Langdon BAC文库为材料,从中筛选鉴定小麦低分子量谷蛋白基因,为深入认识低分子量谷蛋白基因家族的成员组成、分布与进化等特点奠定基础。本研究的主要结果如下:1.利用保守引物对Langdon BAC文库初筛获得的BAC克隆进行PCR二次筛选,鉴定出25个阳性克隆,PCR克隆测序获得26条基因序列,根据推断的氨基酸序列分类原则,它们分属LMW-i、s和m三种类型,其中i型的有9个,s型的有11个,m型的有6个。通过聚类分析,26个基因被分为5类。2.根据不同类型基因序列之间的SNP设计了5对特异引物,特异引物能将不同类型的低分子量麦谷蛋白基因区分开来,并且这些引物在四倍体小麦Langdon及其代换系和六倍体小麦中国春及其缺体-四体中都表现出特异性,26个基因被定位到1A或1B染色体上。3.代表性BAC克隆459F23的测序与分析表明,该BAC克隆全长大约为175kb,含有CACTA、gypsy、copia和LINE等重复序列,一个不表达的低分子量麦谷蛋白基因、抗白粉病基因片段、RFLP标记SFR159,以及一些其它的基因片段。BAC克隆817O07,全长大约为103kb,序列分析表明,该BAC克隆含有CACTA、MITE和gypsy等重复序列,一个M型的低分子量麦谷蛋白基因、HBP-1a转录因子和RFLP标记SFR159。与四倍体小麦Langdon中其他一些BAC序列的微共线性分析表明,低分子量麦谷蛋白基因与RFLP标记SFR159具有很好的共线性。同时,根据SFR159序列的相似性和SFR159与LMW-GS基因之间序列的相似性分析可以推测,BAC817O07和BAC459F23在染色体上相距很近。更多还原

【Abstract】 Low-molecular-weight glutenin subunits (LMW-GS) are a major fraction of wheat glutenin and account for 60% of the glutenins. They play an important role in determining the processing properties of wheat. The LMW-GS are encoded by Glu-A3, Glu-B3 and Glu-D3 on the short arm of chromosome 1A, 1B and 1D. They are present as a multigene family in close association with gliadin. This may lead to the difficulty in studying LMW-GS. With the goal to understand the members of LMW-GS gene family, their location and evolution clearly, Langdon BAC clones from preliminary screening with the Glu-3 coding region as a probe were amplified with conservative primers. Their PCR products were cloned, sequenced and analyzed. Two validated BAC clones were selected to do shotgun sequencing and analysis. The results were as follows:1. With conservative primers, 26 complete gene sequences were obtained by PCR from the 92 BAC clones. With deduced amino acid sequences, they were divided into three types; including 9 i-type, 11 s-type and 6 m-type genes. Phylogenetic tree of nucleotide sequence of this 26 LMW-GS genes showed that they were divided into 5 groups.2. Five pair specific primers were developed based on SNP between different group sequences. These primers can be used to not only identify different type of LMW-GS genes, but also map the genes onto 1A or 1B chromosome using T. durum cv Langdon and its substitution line, Chinese Spring and its nulli-tetrasomic lines.3. Two representative BAC clones were selected to do shotgun sequencing and analysis. BAC459F23 was 173kb. This BAC contained CACTA, gypsy, copia, LINE and other repetitive sequences. It also carried pseudo LMW-GS gene, two powdery mildew resistance gene fragments, RFLP marker SFR159 fragment and some other unknown gene fragments. BAC817O07 was about 103kb. It contained CACTA, MITE, gypsy and other repetitive sequences. It also carried one expressed LMW-GS gene, HBP-1a transcription factor and RFLP marker SFR159 segment. Microcolinearity at Glu-3 loci with different BAC clones in Langdon was revealed. The sequences of SFR159 had high comparability in BAC459F23, BAC817O07, BAC 419P13 and BAC 107G22. The sequence between LMW-GS gene and SFR159 also had high comparability in BAC459F23 and BAC817O07. Therefore, BAC 459F23 and BAC817O07 could be having a close position on chromosome.更多还原