亲缘种质高分子量麦谷蛋白对小麦品质的影响及基因编码区分子克隆

发布时间：2018-05-26 01:22

本文选题：高分子量谷蛋白亚基 + SDS-聚丙烯酰胺凝胶电泳　；参考：《南京农业大学》2016年硕士论文

【摘要】：小麦贮藏蛋白主要由麦谷蛋白和醇溶蛋白组成,谷蛋白由高分子量谷蛋白亚基(HMW-GS)和低分子量谷蛋白亚基组成(LMW-GS)。虽然HMW-GS只占小麦贮藏蛋白总量的10%,但对小麦品质或面筋质量具有决定性的作用。利用特异麦谷蛋白亚基改良小麦品质是小麦育种的重要手段之一。本研究选用携带不同外缘染色体的小麦材料进行研究,鉴定其HMW-GS组成,分析小麦品质,探讨外缘染色体的HMW-GS导入对于小麦加工品质的影响,同时对外缘染色体的HMW-GS基因编码区进行分子克隆,以明确其影响品质的分子基础,从而为小麦品质改良提供参考依据。主要研究内容及研究结果如下:(1)长穗偃麦草代换系和添加系与普通小麦之间籽粒蛋白含量、湿面筋含量、GMP含量和乳酸SRC,以及面团流变学特性均有显著差异。DS1E/1A的面包总分81.5±0.71,显著大于中国春的52±2.83。除DS1E/1D以外,其他长穗偃麦草代换系和添加系,显著增加了面团的形成时间,峰高峰宽,8分钟带宽和乳酸SRC,从而显著增加了面包体积。不同品质指标相关性研究表明,面包体积与GMP含量、乳酸SRC、峰高、峰宽以及八分钟带宽存在显著性相关,与GMP相关系数最高,为0.938。研究还发现碳酸钠SRC与和面时间、峰高、峰宽和八分钟带宽存在显著负相关。因此在早代育种中可以根据GMP含量、乳酸SRC以及峰高等参数的快速测定准确筛选出适合面包加工品质的材料。(2)克隆了长穗偃麦草1E亚基基因序列,该基因CDS序列全长1512个碱基对,推导的氨基酸序列全长为502个氨基酸残基。基因结构与已发表的HMW-GS亚基基因的结构一致,不含内含子,以信号肽、N-端保守区、中央重复区、C-末端构成。与已克隆的HMW-GC基因序列比对,进化树分析将该基因与其他已登录的长穗偃麦草y型基因和中间偃麦草基因聚在一起,与普通小麦的HMW-GS亚基x或y型基因相似度较低,实验材料中的1E基因为y型高分子量麦谷蛋白亚基。(3)对来源于野生粗山草D组染色体的50个人工合成小麦品系进行品质筛选,发现品质性状间存在广泛的遗传变异。以HPLC微量测定谷蛋白大聚体和麦谷蛋白与醇溶蛋白比值为指标,取最高和最低的各3-4份品系进行进一步品质分析,结果表明SE43、SE63和SE76等3个品系在湿面筋含量、籽粒硬度、乳酸SRC及揉混参数等方面与SE32、SE66、SE75和SE77等品系存在显著差异,前3个品系为属强筋硬质,后4个品系属于弱筋软质。(4)克隆了人工合成小麦Glu-D1位点上HMW-GS的x型和y型亚基基因,分析HMW-GS的基因序列和推导的氨基酸序列,结果表明SE43、SE63和SE76等3个品系的Dy亚基基因序列一致,与SE32、SE66、SE75和SE77等品系的Dy亚基基因序列存在较大差异,基因进化树分析,SE63的Dy亚基与拟斯卑尔脱山羊草(A.speltoides)克隆的Dy亚基基因最近,而与粗山羊草中克隆的Dy亚基基因较远。研究HMW-GS的分子结构与小麦面粉品质之间的联系时发现小麦加工品质产生的差异与HMW-GS基因的分子结构有关系,可能是发生β-转角的重复序列和谷氨酰胺残基数量的差异引起的。本研究比较了人工合成小麦2个亚基(Dx和Dy)的中部重复区域中发生β-转角的序列,结果表明,x-亚基含有的四种重复序列多于y-亚基含有的四种重复序列,这表明HMW-GS的x-型亚基的中央重复区含有更多的β-转角结构,对面团加工品质的影响比Y-型亚基要大,预示它们能使面团具有较强的弹性。比较了 2个亚基含有的谷氨酰胺(Q)的数量及其百分含量,结果显示,Dx亚基含有的谷氨酰胺(Q)的数量和摩尔百分含量均比较高,表明在Dx亚基中部重复区域彼此之间以氢键相结合形成长链的能力较强,Dy亚基含有的酪氨酸(Y)的数量较少,但其百分含量较高,达到6.42%。实验中发现品质性状较好的SE63材料在Dx的中部重复序列发生β-转角的序列数量高于品质较差的小麦材料,同样的结论也出现在谷氨酰胺百分比和酪氨酸百分之上,其中筛选出来的SE63材料的谷氨酰胺和酪氨酸百分比分别高达39.91%和6.29%。
[Abstract]:Wheat storage protein is composed mainly of glutenin and gliadin, and glutenin is composed of high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Although HMW-GS only accounts for 10% of the total amount of wheat storage protein, it plays a decisive role in wheat quality or gluten quality. Wheat quality is one of the important means of wheat breeding. In this study, wheat materials with different external chromosomes were selected to study the HMW-GS composition, analyze the quality of wheat, and explore the effect of the HMW-GS introduction of the outer chromosome on the processing quality of wheat, and the molecular cloning of the HMW-GS gene coding region of the external chromophore. The molecular basis of its influence quality was clarifying, thus providing reference for the improvement of wheat quality. The main research contents and results are as follows: (1) the grain protein content, the wet gluten content, the GMP content and the lactic acid SRC, and the rheological properties of the dough have significant differences in the.DS1E/1A surface. The total score of the package was 81.5 + 0.71, which was significantly greater than that of 52 + 2.83. in China's spring, except for DS1E/1D. Other long panicle intermedium and adding lines significantly increased the formation time of the dough, the peak peak width, the 8 minute bandwidth and the lactic acid SRC, thus significantly increasing the volume of bread. The correlation of different quality indexes showed that the volume and GMP content of bread, SRC, There is a significant correlation between peak height, peak width and eight minute bandwidth. The correlation coefficient with GMP is the highest. The 0.938. study also found that there is a significant negative correlation between SRC and surface time, peak height, peak width and eight minute bandwidth. Therefore, in the early generation breeding, it can be accurately selected according to the rapid determination of GMP content, lactic acid SRC and peak parameters. Materials for processing quality of bread. (2) cloned the sequence of 1E subunit gene of Agropyron long panicle, the whole length of the gene CDS sequence was 1512 base pairs. The total length of the deduced amino acid sequence was 502 amino acid residues. The structure of the deduced amino acid was consistent with the published HMW-GS subunit gene, containing no intron, the signal peptide, the N- terminal conservative region, the central repeat area, and the end of C-. Compared with the sequence of the cloned HMW-GC gene, the phylogenetic tree analyses the gene with other logged y and Agropyron intermedium gene and the HMW-GS subunit X or Y gene of common wheat, and the 1E based in the experimental material is derived from the Y type high molecular weight glutenin subunit (3). 50 synthetic wheat lines from the D group of wild wild grasses were selected for quality screening. There were extensive genetic variations among the quality traits. The ratio of glutenin and glutenin to gliadin was measured by HPLC as the index, and the highest and lowest 3-4 strains were taken into one step quality analysis. The results showed that SE43, SE63 and The 3 strains of SE76, such as the content of wet gluten, grain hardness, lactic acid SRC and mixing parameters, were significantly different from those of SE32, SE66, SE75 and SE77. The first 3 strains were strong tendons and the latter 4 belonged to weak tendons. (4) the X and Y subunits of HMW-GS in the Glu-D1 site of the synthetic wheat were cloned, and the HMW-GS gene was analyzed. The sequence and deduced amino acid sequence showed that the sequence of the Dy subunits of the 3 strains of SE43, SE63 and SE76 was the same, and there were great differences with the Dy subunit sequences of SE32, SE66, SE75, SE77 and other strains. The Dy subunit cloned in Aegilops was more distant. The study of the relationship between the molecular structure of HMW-GS and the quality of wheat flour found that the difference in processing quality of wheat was related to the molecular structure of the HMW-GS gene, which may be caused by the difference of the repeated sequence of beta angle and the difference of the number of glutamine residues. The sequence of beta rotation in the middle repeat region of 2 subunits of wheat (Dx and Dy) shows that the four repeat sequences of x- subunits contain more than four repeat sequences containing the y- subunit, which indicates that the central repeat region of the HMW-GS x- subunit contains more beta rotation structure, and the effect of the processing quality of the dough is larger than that of the Y- subunit. The number of glutamine (Q) and its percentage content in the 2 subunits were compared. The results showed that the number of glutamine (Q) and the content of mole percentage of Dx subunits were high, indicating that the ability to form a long chain with hydrogen bonds in the repeating region of the central Dx subunit was stronger, Dy The number of subunits containing tyrosine (Y) is relatively small, but its percentage is higher. The number of SE63 materials with better quality traits in the 6.42%. experiment is higher than that of poor quality wheat materials in the middle repeat sequence of Dx. The same conclusion also appears on the percentage of glutamine and the percentage of tyrosine. The percentages of glutamine and tyrosine of the selected SE63 materials were as high as 39.91% and 6.29%. respectively.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1

【参考文献】