【作者】 徐虹；

【导师】 郭蔼光； 张相岐；

【作者基本信息】 西北农林科技大学 ， 植物学， 2003， 博士

【摘要】 麦谷蛋白是小麦族植物种子中特有的一类贮藏蛋白，在小麦中，是籽粒面筋蛋白的主要成分，与小麦面粉的加工品质密切相关。克隆麦谷蛋白亚基基因，分析麦谷蛋白的分子特征，对研究麦谷蛋白亚基的功能、利用优质亚基进行品质改良具有重要的意义。而研究小麦近缘种属的麦谷蛋白，有助于人们得到更多的亚基变异类型，筛选到更多的有用基因。 中间偃麦草（Thinopyron intermedium 2n=6X=42，E₁E₁E₂E₂XX）是小麦的一个近缘种，含有大量可用于小麦品种选育的有益性状。从二十世纪30年代开始，人们就进行了普通小麦与中间偃麦草的杂交工作，并已将许多中间偃麦草的有用基因导入小麦中，获得了丰富的遗传种质资源。 本实验以中间偃麦草，小麦-中间偃麦草部分双二倍体、小麦-中间偃麦草二体异附加系为材料，初步研究了中间偃麦草麦谷蛋白亚基组成、再次鉴定中间偃麦草的特异麦谷蛋白亚基，克隆了中间偃麦草麦谷蛋白基因、并分析基因的结构特征，为在中间偃麦草中筛选、鉴定可用于小麦品质改良的优质基因奠定基础。 经过研究，主要获得以下结果： 1 本文通过种子麦谷蛋白SDS-PAGE图谱分析，初步研究了中间偃麦草的麦谷蛋白亚基组成，并分析其与小麦麦谷蛋白亚基谱带的差别，揭示了中间偃麦草麦谷蛋白基因位点与小麦基因位点之间存在差异。 2 采用Genomic PCR，用简并性引物从中间偃麦草中克隆到6个高分子量麦谷蛋白基因Ax-1、Ax-3、Ay-1、Ay-2、Ay-3、Ay-4，基因结构分析表明，它们与已经发表的分子量麦谷蛋白基因具有类似的一级结构特征。通过与小麦和其它物种的基因序列进行比较和聚类分析，表明它们分别与x-型和y-型亚基高度同源。4个y-型亚基基因序列中出现了链内终止密码子，是不能表达的假基因。广型亚基基因Ay-1与Ay-3的N-末端序列具有105个氨基酸，与已经报道的其它y-型亚基基因不同，在这两个基因的N-末端保守序列区存在较多的氨基酸变异位点。 3用Genomic PCR在中间偃麦草中克隆到4个有活性的低分子量麦谷蛋白基因，并分析了基因的结构特征。发现，中间偃麦草中存在两种新的低分子量麦谷蛋白变异类型，分别命名为Ai-M型与Ai-Q型亚基。pAL506、pAL507和pAL1306编码多肽的N-末端序列是MESNIIISFL-，属于Ai-M型；pAL1231编码亚基序列中缺失N-末端，信号肽后紧接重复序列QQQLPQQ-，属于Ai-Q型。通过基因序列的聚类分析表n摘要明，4个新基因与小麦的低分子量麦谷蛋白基因具有80%的同源性. 4采用SDS一队GE和Genomic PCR相结合的方法在小冰麦异附加系TAI系列中鉴定出异附加系TAI一13和TAI一25中含有中间惬麦草特异麦谷蛋白亚基，附加染色体上具有麦谷蛋白基因位点，附加染色体属于中间僵麦草第一同源群.并发现，在异附加系TAI一11中也具有中间僵麦草低分子量麦谷蛋白基因位点，但对附加染色体所属同源群还不能确定. 5用RT一PCR的方法从TAI一13未成熟籽粒中克隆到4个来自中间僵麦草的低分子量麦谷蛋白基因，分别属于Ai一M型、Ai一Q型和Ai一I型.Ai一I型基因的N一末端缺失，信号肤后紧接重复序列ISQQQ一，是小麦中已经报道的基因类型。其中一个Ai一M型基因1 3514编码氨基酸序列中含有9个Cys位点，中间重复区具有26个重复模块，具有比较特殊的分子结构，值得进一步研究. 6两个Ai一M型中间僵麦草低分子量麦谷蛋白基因（pAL507和pALI 306）在大肠杆菌中得到表达，根据SDS一队GE相对迁移率计算，相对分子量分别是38，600和39，500，进一步研究亚基的功能提供了可能性 7通过对所克隆的中间僵麦草麦谷蛋白基因的序列特征分析，证实了中间僵麦草中存在不同于小麦的麦谷蛋白亚基，可以作为小麦品质育种的一个新的种质材料.麦谷蛋白基因位点可以作为生化标记和分子生物学标记应用于遗传学研究.更多还原

【Abstract】 Glutenin is the special storage protein in seed of Triticeae, play an significant role in the determination of flour processing quality as the primary component of gluten protein of wheat grain. For the study of subunit function and quality breeding of wheat with good quality gene , it’s important to clone the glutenin subunit gene and assess the molecular characters of glutenin. The information of glutenin from the wheat relative speices would be helpful to obtain more varieties of glutenin subunit and screen the useful gene for wheat improvement.Thinopyrum intermedium (Host) Barkwarth (= Agropyron f intermedium (Host)Beav or Elytrigia intermedia (Host) Nevski, 2n=6X=42, EjE^EiXX), is one of wheat related species, possess many desirable characteristics for wheat improvement. Since 1920s, a number of wheat Th. intermedium crossing groups have been done and a series of hybrids have been developed, many useful genes of Th .intermedium have been transferred to wheat.In present paper, Th .intermedium, T. aestivum-Th. intermedium partial amphiploids , wheat- disomic alien addition lines (TAI- I &TAI- II series) were used to study the constitution of glutenin subunit of Th. intermedium , identify the speciality subunits of Th. Intermedium, clone the glutenin subunit genes from Th. Intermedium, and analyze the molecular characterization of Th. intermedium genes. It would be essential research to find candidate genes from Th. intermedium for wheat quality breeding.The important results obtained as followings .1 We analyzed the glutenin SDS-PAGE profiles and studied the constitution of glutenin subunits of Th. Intermedium. By the comparison with wheat glutenin subunit bands, the difference between glutenin locus of wheat and Th. Intermedium were described.2 Six High-molecular-weight glutenin subunit genes of Th. Intermedium were cloned by Genomic PCR with a parr of degenerate primers. These genes were named as Ax-1, Ax-3, Ay-1, Ay-2, Ay-3 and Ay-4. They had similar primary structure with other reported HMW-GS genes, were high degree homology with x-type and y-type subunit genes of wheat respectively. There were in-frame stop codon in 4 novel y-type subunitgenes, indicated they were pseudogenes. The N-terminus of Ay-1 and Ay-3 contained 105 amino acid residues and had many mutation points, different from other y-type genes.3 Four novel Low-molecular -weight glutenin subunit genes have been cloned from genomic DNA of Thinopyrum intermedium. According to N-terminus sequence of amino acid deduced from nucleotide, these genes could be classified into two new types termed as Ai-M and Ai-Q type LMW-GS gene. The alignment of primary structure suggested four genes were different from previous published LMW-GS genes. Three of these pAL1306, pAL506 and pAL507 fitted into Ai-M type, which encoded polypeptides with N-terminal sequences of MESNIIISFLKPWL-. The pAL1231 encoded a Ai-Q type subunit, mature peptide had a N-terminal deletion, signal peptide followed by amino acids sequence of QQQLPQQ-.4 The SDS-PAGE and genomic PCR have been employed to characterize the glutenin and glutenin subunit locus of Triticum aestivwn-Thinopyrum intermedium alien addition series. The results indicated that the addition lines TAI-13 and TAI-25 carried a pair of Th. intermedium chromosomes with glutenin subunits gene loci, and the extra chromosomes belonged to the homoeologous group 1. The addition line TAI-11 contained the low-molecular -weight glutenin subunit gene loci, but the homoeologous of extra chromosome couldn’t been determined.5 Then, four low-molecular-weight glutenin subunit (LMW-GS) genes (13003, 13006, 13045 and 13514) originated from Th. intermedium were isolated from immature endosperm of TAI-13 by RT-PCR. They belonged Ai-M type, Ai-Q type , and Ai-I type subunit genes respectively. The polypeptids coded by 13514 had the specific primary structure, which contained 9 Cys residue in molecular, and 26 repeat units, could formed 3 inter-molecular disulphide bonds by inference, and wa更多还原