【作者】 房敬业；

【导师】 何光源； 杨广笑；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 小麦高分子量麦谷蛋白亚基(high molecular weight glutenin,HMW-GS)是籽粒贮藏蛋白的重要组分。HMW-GS的组成和含量直接影响和决定着小麦的加工品质。粗山羊草(Aegilops tauschii,DD,2n=2x=14)是普通小麦六倍体的D基因组的供体,存在丰富的HMW-GS的变异类型,且大多是现有普通小麦中不存在的新类型,是普通小麦品质改良的一个重要基因资源。因此从粗山羊草中分离克隆这些新的HMW-GS基因,不仅为粗山羊草与普通小麦的进化关系提供更多的遗传信息,而且能够为普通小麦品质改良提供基因资源并拓宽普通小麦的遗传多样性。本文以粗山羊草为材料,对三个新的HMW-GS基因进行了克隆、序列比较和系统进化分析,并对1Dx1.1~t基因进行了原核表达和基因枪转化。主要研究结果如下:(1)一个新的x-型HMW-GS基因的分子克隆、原核表达及其系统进化分析通过对山羊草种子储藏蛋SDS-PAGE电泳分析发现了一个新的HMW-GS,其在迁移率比亚基1Ax1的稍慢,我们将其命名为1Dx1.1~t。利用等位基因特异性PCR(Allele specific PCR,AS-PCR)方法对其基因编码区进行了分离。1Dx1.1~t基因全长为2628 bp,编码874个氨基酸,是目前山羊草中分子量最大的HMW-GS基因。原核表达结果表明1Dx1.1~t基因表达蛋白与种子中相应的谷蛋白亚基迁移率相同,这说明所克隆基因确实是1Dx1.1~t亚基的编码基因。序列比较结果表明1Dx1.1~t与其它Dx型亚基高度相似,但也具有其它Dx型亚基不具有的新颖特征。首先,在其它Dx型亚基的N-末端都有的一个保守的三肽序列(LQE),而在1Dx1.1~t的相应的区域是不存在的;其次,在1Dx1.1~t中部重复区域有3个三肽重复的结构域(GQQGQQ)以及一个新的三肽(GQL)。系统分析结果表明亚基1Dx1.1~t与其它Dx型亚基优先地聚在一起和至少有三种基因型的粗山羊草参与普通小麦的形成。(2)两个新的x-型HMW-GS基因的分离、二级结构预测及其系统进化分析利用AS-PCR法方法克隆了两个新的y-型HMW-GS基因,分别命名为1Dy12.5~t和1Dy9.5~t。1Dy12.5~t基因编码的蛋白在SDS-PAGE上的电泳迁移率远快于亚基1Dy12,而1Dy9.5~t基因编码的蛋白的电泳迁移率处于亚基1Dy10和1By9之间。1Dy12.5~t基因全长为2650 bp,其中编码区长度为1788 bp和上游区域长度为862 bp;1Dy9.5~t基因编码区长度为1995 bp,是目前分子量最大的Dy型亚基基因。y-型HMW-GS基因上游区域的核苷酸序列比较结果1Dy12.5~t基因具有其它y-型HMW-GS基因上游区域的保守区域,显示其是一个有转录活性的基因,但在其启动子(ATG)与CCACC序列之间有一个5 bp(ACGAG)的插入,这个5 bp的插入是在已知的HMW-GS基因中所没有发现的。亚基1Dy12.5~t和1Dy9.5~t具有典型y型亚基的结构特征。亚基1Dy12.5~t与1Dy13~t高度相似只有9个氨基酸残基的差别;而1Dy9.5~t与1Dy12.4~t和1Dy高度相似,分别有2和4个氨基酸残基的差别。通过比较1Dy9.5~t与1Dy12.4~t基因序列发现可能是由于在1Dy9.5~t基因第806和1478位的两个16 bp核苷酸正向重复引起224个氨基酸残基的缺失而导致1Dy12.4~t基因的产生。在1Dy12.5~t基因编码区内分别检测到5个SNPs,其中3个SNPs引起氨基酸残基的改变;在1Dy9.5~t基因编码区内分别检测到3个SNPs,其中2个SNPs引起氨基酸残基的改变。蛋白质二级结构预测亚基1Dy9.5~t可能是一个优质亚基,而1Dy12.5~t可能是一个劣质亚基。系统分析表明至少有两种基因型的粗山羊草参与普通小麦的形成和粗山羊草还参与柱穗山羊草的形成。(3)1Dx1.1~t基因的基因枪转化构建了1Dx1.1~t基因的转基因表达载体,对其进行了基因枪转化,为进一步鉴定其对小麦加工品质的影响奠定了基础。更多还原

【Abstract】 High molecular weight glutenin subunits(HMW-GS) are one group of the major components of storage proteins in wheat seeds.Variation in the amount and composition of HMW-GS would affect and determine the flour processing quality.Aegilops tauschii (DD,2n=2x=14,syn.Ae.squarrosa) is the D genome donor of hexaploid wheat. Extensive allelic variation occurs in HMW-GS composition in Ae.tauschii more than in the subunits encoded by the D genome of modern wheat cultivars,and many new allelic variation does not exist in modern bread wheat.Therefore,cloning HMW-GS genes from Ae.tauschii would not only provide useful resources for wheat quality improvement by increasing the genetic diversity of the D genome,but also offer more genetic information on the evolutionary relativeship between bread wheat and Ae.tauschii.The objectives of this work were to isolate and characterize novel HMW-GS genes from Ae.tauschii.By comparing their deduced amino acid sequences with these previously characterized Dx or Dy type HMW-GS,it can help us to understand the evolution of Glu-D1 alleles.Besides, in order to investigate effect of 1Dx1.1~t on flour processing quality,1Dx1.1~t gene was transferred into E.coil and wheat.The main results are as follows:(1) Molecular cloning,bacterial expression and phylogenetic analysis of a novel x-type HMW-GS geneA novel x-type HMW-GS possessing a slightly slower mobility than that of subunit 1Ax1 in SDS-PAGE,which was designated 1Dx1.1~t in Ae.tauschii was identified.Its gene was amplified with allele-specific PCR(AS-PCR) primers.The open reading frame (ORF) of the gene was 2628 bp,encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1~t had high similarity with other 1Dx subunits but has two unique characteristics.Firstly,a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits is absent from subunit 1Dx1.1~t.Secondly,three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence(GQL) are present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1~t preferentially clustered with other known 1Dx subunits and at least three Ae.tauschii genotypes may have been involved in the polyploidization process during the origin and evolution of bread wheat.(2) Isolation,secondary structure predicton and phylogenetic analysis of two novel y-type HMW-GS genes Two novel y-type HMW-GS genes were cloned with AS-PCR method,designated 1Dy12.5~t and 1Dy9.5~t in Ae.tauschii,respectively.1Dy12.5~t encoded a protein that has much faster mobility than that of subunit 1Dy12 in SDS-PAGE.1Dy9.5~t encoded protein that has faster mobility than that of subunit 1By9,but slower mobility than that of subunit 1Dy10 in SDS-PAGE.A nucleotide sequence of 2,650 bp containing an open reading frame of 1,788 bp and upstream sequence of 862 bp was obtained.Nucleotide sequence comparisons showed that the 5’flanking region shared the conservative domains of y-type HMW-GS gene promoters,indicative of its transcriptional activity.However,there was a 5 bp(ACGAG) insertion before ATG(start codon) in 1Dy12.5~t subunit gene,which had not been observed in other known HMW-GS genes.1Dy9.5~t and 1Dy12.5~t had the typical primary structure of y-type HMW-GS.The deduced amino acid sequence of subunit 1Dy12.5~t showed greater similarity to subunit 1Dy13~t,differing by only nine amino acid residues.The deduced amino acid sequence of subunit 1Dy9.5~t displayed greater similarity to subunit 1Dy12.4~t and 1Dy,differing by only two and four amino acid residues, respectively.Compared the nucleotide acid sequence of subunit 1Dy9.5~t with that of subunit 1Dy12.4~t,we found that two 16 bp direct repeats(DR) existed in sites 806 and 1478 in the 1Dy9.5~t subunit gene,resectively,and inferred that these two 16 bp DR could make 224 amino acid residues deletion of 1Dy9.5~t which made 1Dy9.5~t to 1Dy 12.4~t.Five single nucleotide polymorphisms(SNPs) were detected in the coding region of the subunit 1Dy12.5~t gene,three of which resulted in the residue substitutions.Three SNPs were detected in the coding region of the subunit 1Dy9.5~t gene,among which two SNPs resulted in residue substitutions.Secondary structure prediction results showed that subunit 1Dy9.5~t could have a good bread processing quality while subunit 1Dy12.5~t could have a poor bread processing quality.Phylogenetic analysis showed that at least two genotypes of Ae.tauschii might be involved in the formation of bread wheat and Ae. tauschii could be also be involved in the formation ofAegilops cylindrica.(3) Transformation 1Dx1.1~t gene into wheat by particle bombardmentWe constructed the transgene expression vector of 1Dx1.1~t gene and transformed this vector into wheat by particle bombardment,which provided with the basis for further analyzing the function of this gene in influnceing flour processing quality.更多还原