【作者】 张艳贞；

【导师】 晏月明；

【作者基本信息】 首都师范大学 ， 遗传学， 2007， 博士

【摘要】 小麦近缘种——粗山羊草（Aegilops tauschii，2n=2x=14，D^tD^t）是普通小麦D基因组供体，存在广泛的高分子量谷蛋白亚基（HMW-GS）等位变异，是小麦品质改良的重要基因资源。分离克隆这些基因不仅可以为小麦的品质育种提供具有自主知识产权的基因资源、拓展遗传基础，而且有望为研究六倍体小麦的起源和进化提供有用的信息和证据。本研究从粗山羊草中分离鉴定了3个新的HMW-GS基因，并进行了异源表达、系统进化分析、翻译后修饰鉴定以及植物双元表达载体构建和烟草转化，主要结果如下：1．粗山羊草HMW谷蛋白新亚基基因的分离与原核表达以粗山羊草TD81和TD130为材料，通过SDS-PAGE、HPCE、RP-HPLC和MALDI-TOF-MS鉴定了两对新的Glu-1D HMW谷蛋白亚基组合，分别命名为1Dx5^*t+1Dy10.1^t和1Dx5.1^*t+1Dy12.1^*t。采用AS-PCR方法，分离克隆了1Dx5^*t、1Dx5.1^*t和1Dy12.1^*t亚基的编码基因，分别为2487bp、2532bp和1947bp，编码827、842和648个氨基酸。DNA序列及推导氨基酸序列具有典型的HMW-GS的序列结构特征，而且1Dx5^*t亚基重复单元更接近一致序列，相对变异率较低，主要体现在每一重复单元的平均变异数和重复单元发生变异的频率上。1Dx5^*t和1Dx5.1^*t推导蛋白分子量与质谱测定分子量非常一致，而1Dy12.1^*t推导蛋白分子量与质谱测定分子量则相差很大，远远超过误差允许范围。设计2对不含信号肽编码区的引物，分别特异扩增基因1Dx5^*t、1Dx5.1^*t和1Dy12.1^*t，然后与原核表达载体pET30a相连，三个基因在大肠杆菌BL21（DE3）plysS中均得到了高效表达。SDS-PAGE分析表明，基因1Dx5^*t和1Dx5.1^*t表达蛋白与种子中相应的谷蛋白亚基迁移率相同，确证所克隆基因确实为所鉴定新亚基的编码基因。但同时发现几个含有1Dx5^*t的重组质粒表达出一个迁移率超前的小蛋白，而且N-端测序表明大肠杆菌中表达蛋白与相应种子蛋白氨基酸序列相同。这几个重组质粒的测序结果显示，在大肠杆菌中1Dx5^*t基因内发生了180bp的缺失和一个提前终止密码子的突变，由变异了的基因序列推导的蛋白分子量与表达的小蛋白在SDS-PAGE上的迁移率也非常相符。这一结果首次提供了谷蛋白亚基编码基因重组克隆发生变异的直接证据，变异发生的分子机制可能是由于链内不合理重组（illegitimate recombination）的发生，这种机制很可能对谷蛋白亚基基因新的等位变异的产生发挥重要作用。而1Dy12.1^*t基因序列原核表达结果则显示表达蛋白较种子内相应亚基SDS-PAGE迁移率稍快。2．Glu-1D谷蛋白亚基编码基因系统进化分析分别采用Network分析、neighbor-joining tree和同源树构建对Glu-1D-1和Glu-1D-2位点等位变异间的系统进化关系进行分析，结果表明1Dx5^*t，1Dx2^t，1Dx1.6^t真和1Dx2.2^*均处于源节点，代表比较原始的基因序列，这暗示六倍体小麦至少起源于4个独立的杂交事件。Network、neighbor-joining tree和同源树分析结果相互吻合，表明普通六倍体小麦D基因组是多起源的。结合多起源假说与不合理重组的分子机制，提出了Glu-1D-1位点等位变异可能的产生途径：9个已克隆的Dx亚基基因由1Dx5^*t，1Dx2^t，1Dx1.6^t和1Dx2.2^*分化而来。特别地，1Dx2.2^*基因可能早先就存在于粗山羊草中（也可能由粗山羊草中一个更原始的基因衍生而来），在某一时期通过某种罕见的远交或异型杂交转移到普通小麦中。由于1Dx2.2^*基因内561bp的长正向重复序列可能发生不合理重组，产生了类似1Dx2基因，该基因在小麦进化过程中通过点突变、非均等交换等事件，进而产生了1Dx2.2基因。3．谷蛋白亚基1Dy12.1^*t翻译后修饰分析鉴于1Dy12.1^*t亚基质谱测定分子量与基因推导蛋白分子量的巨大差异和SDS-PAGE的反常电泳现象，采用液质联用技术对其进行了蛋白翻译后修饰的鉴定。理论上1Dy12.1^*t亚基含有13个精氨酸和7个赖氨酸，且精氨酸和赖氨酸均不与脯氨酸相连，经Trypsin充分酶解后，应该产生21个不同的肽段，实际鉴定唯一肽段数是12个，蛋白鉴定覆盖率最高19.8％。虽然离国际认可的蛋白覆盖率还有一定差距，但从1Dy12.1^*t亚基翻译后修饰位点预测结果看，很多可能性高的修饰位点所在的肽段可能还没有得到很好的鉴定，所以仍不能排除该亚基存在某种程度的翻译后修饰（如糖基化和磷酸化等）的可能。而且我们发现另一材料TD87中较1Dy12.1^*t亚基SDS-PAGE迁移率稍快、MALDI-TOF-MS测定分子量相差很大的1Dy亚基，其编码基因序列却与1Dy12.1^*t完全相同；又考虑该基因序列原核细胞表达蛋白较种子蛋白1Dy12.1^*t亚基迁移率稍快、烟草种子胚乳表达蛋白与1Dy12.1^*t亚基迁移率一致、免疫反应性相同的事实，我们认为TD130中1Dy12.1^*t亚基存在翻译后修饰的可能性较大，进一步的鉴定还在进行中。4．谷蛋白基因植物双元表达载体的构建及其烟草转化针对克隆基因DNA序列特点，构建了两种植物双元表达载体pBI121PVP1-Glu和pBINHP-GluT，经一系列中间载体和最终载体的酶切鉴定，证明表达载体的构建完全正确。载体pBINHP-GluT的构建，精心选择经试验验证了其驱动活性的胚乳特异性启动子，选择添加HMW谷蛋白亚基基因和大部分LMW谷蛋白亚基因内部均不含有的XbaⅠ和KpnⅠ内切酶位点，并将目的谷蛋白亚基编码基因的调控置于T-DNA区内，从而使这一载体在植物遗传转化中具有很大的优势。根癌农杆菌介导烟草叶盘转化结果表明，1Dy12.1^*t基因在烟草种子胚乳中得到成功表达，表达蛋白与TD130相应亚基具有相同的SDS-PAGE迁移率，且HMW-GS多克隆抗体Westhern blotting检测为阳性，不仅证明所克隆1Dy12.1^*t亚基编码基因的正确性，而且验证了所构建载体的有效性，为下一步小麦的遗传转化奠定了基础。更多还原

【Abstract】 The wheat storage proteins, especially the compositions and content of high molecular weight glutenin subunits （HMW-GS） play important roles in the flour processing and bread-making quality. Previous investigations showed that the genetic foundation of hexaploid wheat was limited whereas there were extensive allelic variations of HMW-GS in wheat related species, such as Aegilops tauschii. Therefore, molecular cloning and phylogenetic analysis of novel HMW glutenin subunit genes from wheat related species would not only provide potential elite gene resources for wheat quality improvement, but also offer useful information and evidence for further understanding the origin and evolution of hexaploid wheat. This work included gene cloning, heterogenous expression, post-translational modifications, phologenetic analysis of novel Glu-1 allelles from Aegilops tauschii, plant binary expression vector construction and transformation of tobacco with cloned glutenin subunits genes. The main results obtained were as followings:1. Isolation and heterogenous expression of novel HMW glutenin subunit genes in Aegilops tauschiiTwo pairs of novel HMW glutenin subunits in Aegilops tauschii （2n=2x=14, D^tD^t） TD81 and TD130 were identified by SDS-PAGE, RP-HPLC and MALDI-TOF-MS and designated as 1Dx5^*t +1Dy10.1^t and 1Dx5.1^*t + 1Dy12.1^*t, respectively. The coding genes of 1Dx5^*t, 10x5.1^*t, and 1Dy12.1^*t were isolated by AS-PCR and the complete ORFs were obtained. The 1Dx5^*t consists of 248lbp encoding a mature protein of 827 residues with the deduced M_r of 85,782Da while 1Dx5.1^*t comprises 2526bp encoding 842 residues with 87,663Da, and 1Dyl2.1^*t comprises 1947bp encoding 648 residues with 67,347.6Da. The nucleotide acid sequences and deduced amino acid sequences had typical characters of HMW glutenin subunits. The average numbers of variants per repeat unit and the mean frequency of variant units of subunit 1Dx5^*t were relatively lower, indicating that its repeat units was closer to the consensus.The deduced M_r^s of mature proteins encoded by 1Dx5⁹（*t） and 1Dx5.1^*t were consistent with those determined by MALDI-TOF-MS, while the deduced M_r of 1Dy 12.1^*t was much lower than that determined by MALDI-TOF-MS. Three novel genes Wx5^*t, Wx5.1^*t and IDy 12.1^*t had been submitted to GenBank with accession numbers DQ681076, DQ681077 and DQ681079. Two pairs of differential primers were designed containing no signal encoding domain to amplify Wx5（*t）, Wx5.1^*t and 1Dy 12.1^*’ genes, the PCR products were cloned into the bacterial expression vector pET30a and the hybrid vectors were transformed into E. coli strain BL21 （DE3）pLysS. Expression of the two novel ORFs of 1Dx5^*t and 1Dx5.1^*t and N-terminal sequencing confirmed the authenticities of the two novel genes. Interestingly, several hybrid clones of 1Dx5^*’ gene expressed an ilk smaller protein relative to the authentic subunit present in seed proteins, which was confirmed to resulted from a long deletion of 180bp through illegitimate recombination as well as a in-frame stop codon formation. The derived molecular weight of the small protein was well consistent with the result of SDS-PAGE analysis. Our investigations provided the direct evidence for variable recombinant clones and brought out the possible mechanism of illegitimate recombination for this variation, which may provide useful information for further insights into glutenin gene variations and their evolutionary direction. It was interesting that the expressed protein of 1DyH.1^*t gene ORP moved slightly faster than lDy12.1^*t subunit from TD130 on SDS-PAGE.2. Phylogenetic analysis of Glu-1D allellesThe network, neighbor-joining tree and homological free were constructed to analyse the phylogenetic evolution of Glu-1D alleles. The results demonstrated that the 1Dx5^*t, 1Dx2^t, 1Dx1.6^t and 1Dx2.2^* represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in the associated star-like phylogeny. The results of network, neighbor-joining tree and homological tree analysis happened to have the same view, which suggested that there were more than one independent origins of hexaploid wheats. A possibly evolutionary way of Glu-D-1-1 alleles could be drawn from our analysises: the 1Dx2.2^* , deriving from a more primitive gene, was present in Ae. tauschii, and then transferred by rare outcrossing into common wheat. A mutational event, probably by illegitimate recombination, resulted in a large DNA fragment loss of 1Dx2.2^* and the generation of 1Dx2 gene. In certain recent evolutionary time of common wheat, a large fragment duplication in the repetitive domain of 1Dx2, presumably resulting from unequal crossing over as well as illegitimate recombination, was responsible for generating the 1Dx2.2 gene.3. Identification of post-translational modifications （PTMs） of 1Dy12.1^*t subunitBecause of the evident difference between the deduced M_rs and that determined by MALDI-TOF-MS and the anomalous electrophoretic behavior on SDS-PAGE, the reversed-phase high-performance liquid chromatography following by electrospray ionization mass spectrometry （RP-HPLC/ESI-MS） were performed to explore the post-translational modifications in 1Dy12.1^*t subunit. In theory, 1Dy12.1^*t subunit contained 13 Arg and 7 Lysresidues, and absence of Arg-Pro bonds, thus the production of 21 tryptic peptides was expected. The ESI-MS analysis of 1Dy12.1^*t tryptic digest resulted in the identification of 12 incomplete unique peptides, which was less than 50% of that in theory. Although the results gave the highest sequence coverage of 19.8%, less than the accepted coverage, the certain post-translational modifications such as glycosylation and phosphorylations can not be ruled out. Moreover, the 1Dy subunit of another accession TD87, which moved slightly faster on SDS-PAGE and had a largely smaller M_rs determined by MALDI-TOF-MS than 1Dy12.1^*t subunit, had the absolutely same encoding sequeces as 1Dy 12.1^*t gene. We also found that although the expressed protein of 1D12.1^*t gene ORF in E coli moved slightly faster than 1Dy12.1^*t subunit from TD130, the expressed protein of the gene in tobacco endosperm had the same mobility and immunity as 1Dy12.1^*t subunit from TD130. So we speculated that certain post-translational modifications were most possibly exsisted in 1Dy12.1^*t subunit of TD130.4. Construction of plant binary expression vector containing glutenin subunit genes and transformation of tobaccoTwo kinds of plant binary expression vectors pBI121PVP1-Glu and pBINHP-GluT were constructed based on analysis of nucleotide acid sequences character. A series of identification of midterm and final vectors by suitable different restriction enzymes showed that the vectors construction was absolutely successful and correct. Especially the vector pBINHP-GluT, which placed the target gene under the regulation of the verified endosperm-specific promoter, which appended the restriction sites XbaI and KpnI beside 5’ and 3’ of the target glutenin subunit gene, the XbaI and KpnI sites have not been found in all published HMW-GS genes and most LMW-GS genes, furthermore, which placed the all operation domains between the left and right borders of T-DNA, had great predominance in plant transformation. The results of transformation of tobacco lamina mediated by Agrobacterium tumefaciens showed that in the endosperm of tobacco gene 1Dy 12.1^*t expressed a protein which had similar mobility with 1Dy12.1^*t subunit extracted from TD130, furthermore, westhern blotting showed that the expressed protein and extracted 1Dy12.1^*t subunit from TD130 all strongly hybridize with the multiclonal antibodies against HMW glutenin subunits, which confirmed the authenticities of the novel gene 1Dy 12.1^*t and the validity of the plant binary expression vector.更多还原