小麦三个贮藏蛋白基因的克

发布时间：2018-06-05 21:18

本文选题：普通小麦 + 高分子量谷蛋白　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：小麦贮藏蛋白分两种:麦谷蛋白(Glutenin)和醇溶蛋白(Gliadin),麦谷蛋白也分两种:高分子量谷蛋白亚基(high molecular weight glutenin subunit,HMW-GS)和低分子量谷蛋白亚基(low molecular weight glutenin subunit,LMW-GS)。小麦贮藏蛋白是小麦面粉加工品质的决定性因素,其含量超过小麦籽粒蛋白总量的80%。本研究以课题组自主选育的优质小麦新品种“西农538”和“西农558”的基因组DNA为模板,根据GenBank中收录的HMW-GS、LMW-GS和α-gliadin的基因序列,设计特异性引物,通过PCR扩增、克隆测序等实验技术,分别从这两个小麦品种中得到了三种蛋白的基因编码序列各一条,并进一步进行了序列比对及进化树分析,同时以“西农538”中获得的三条基因序列为目标序列,诱导其在大肠杆菌表达系统中表达,然后通过蛋白纯化和微量掺粉实验研究比较这三个表达蛋白各自对小麦面粉的品质效应。实验结果如下:1.利用设计的特异性引物,共得到了6条具有单一完整开放阅读框的基因序列。其中从“西农538”中克隆得到的HMW-GS、LMW-GS和α-gliadin基因序列的GenBank登录号分别为KX452083、KX452081、KX452085,编码区长度分别为1812 bp、915 bp和897 bp;从“西农558”中克隆得到的这三种蛋白的基因序列的登录号分别为KX452084、KX452082、KX452086,编码区长度分别为1977 bp、915 bp和879 bp。2.通过氨基酸序列对比发现:KX452083和KX452084主要由信号肽、N-端保守区、中间重复区和C-末端四个部分构成,都有7个半胱氨酸(Cys)残基,中间重复区有六肽和九肽重复单元,但没有三肽重复单元,由此推测这两个基因序列可能属于y型亚基,同时经过blastp比对和进化树分析得知,两条序列分别与1Dy10和1Dy12亚基有极高的相似度。KX452081和KX452082主要由信号肽、N-端保守区、中间重复区(该区域是谷氨酰胺(Q)和脯氨酸(P)的富集区)、C-端保守区Ⅰ(含有5个Cys残基)、C-端保守区Ⅱ(该区域含有丰富的谷氨酰胺(Q)和1个Cys残基)和C-端保守区Ⅲ(含有1个Cys残基)这六部分组成,通过序列比对、N/C端氨基酸组成(METRCIPG······VGTGVGAY)、Cys残基的位置和进化树分析发现,得到的这两条LMW-GS序列属于Glu-D3位点、Type V(Group 10)、m型、C组LMW-GS。KX452085和KX452086主要由6部分组成:信号肽、N-端重复区、多聚谷氨酰胺区Ⅰ、特征区Ⅰ、多聚谷氨酰胺区Ⅱ和特征区Ⅱ,其中KX452085含有6个Cys残基,可形成3对分子内二硫键;KX452086含有7个Cys残基,不仅可以形成3对分子内二硫键,多余的一个Cys残基可能形成一个分子间二硫键。3.依据本实验的研究目的和前人的研究成果,以“西农538”中克隆得到的三条序列(KX452083、KX452081和KX452085)为研究对象,经IPTG诱导,使它们在宿主菌Escherichia coli Trans B(DE3)中进行体外表达,通过SDS-PAGE凝胶电泳和Western blot检测鉴定,结果表明这三个蛋白均成功表达。4.对三个蛋白进行大量的体外表达,经回收纯化、低温冷冻干燥和微量掺粉实验(以中国春面粉为基础面粉),得到了一系列相关参数。分析这些参数发现,KX452083(HMW-GS)与面粉筋力正相关,但对面团弹性产生了负效应,并且不利于面团的延伸;KX452081(LMW-GS)对面粉筋力的影响比较复杂,尚难确定相关性,但其对面粉弹性有增强作用,而对面团的延伸不利;KX452085(α-gliadin)对面粉筋力有负效应,但使面团更易流变,且对面团的延伸和弹性有显著的改善。此外,粉质质量指数(Farinograph quality number,FQN)是一种被用来评价面粉质量的常用指标,以FQN为衡量标准分析比较这三个蛋白的品质效应,结果为:HMW-GSα-gliadinLMW-GS。
[Abstract]:Wheat storage protein is divided into two kinds: Wheat Glutenin (Glutenin) and gliadin (Gliadin), and glutenin is also divided into two kinds: high molecular weight glutenin subunit (high molecular weight glutenin subunit, HMW-GS) and low molecular weight glutenin subunit (low molecular weight glutenin). Wheat storage protein is the quality of Wheat flour processing. The determinant factor, its content exceeded the total amount of wheat grain protein 80%., based on the genome DNA of "Xi Nong 538" and "Xi Nong 558", a new high quality wheat variety selected by the project group, according to the gene sequence of HMW-GS, LMW-GS and alpha -gliadin included in GenBank, specific primers were set up and amplified by PCR, cloned and sequenced. In the experimental technique, one of the gene encoding sequences of three proteins was obtained from these two wheat varieties, and the sequence alignment and evolutionary tree analysis were further carried out. At the same time, three gene sequences obtained in "Xi Nong 538" were used as the target sequence to induce them to be expressed in the Escherichia coli system, and then purified and trace by protein. The effect of the three expressed proteins on wheat flour quality was compared. The experimental results were as follows: 1. using the specific primers designed, 6 gene sequences with single complete open reading frame were obtained. The GenBank login number of HMW-GS, LMW-GS and alpha -gliadin gene sequences cloned from "Xi Nong 538" KX452083, KX452081, KX452085, the length of the coding region were 1812 BP, 915 BP and 897 BP, respectively. The sequence numbers of the three proteins from "Xi Nong 558" were respectively KX452084, KX452082, KX452086, the length of the coding region was 1977 BP, 915 BP and 879 bp.2. were compared with the amino acid sequence. 84 mainly consists of signal peptide, N- terminal conservative region, intermediate repeat area and four parts of C- terminal, 7 cysteine (Cys) residues, six peptide and nine peptide repeating units in the middle repeat area, but no three peptide repeating unit, thus speculating that the two gene sequences may belong to y subunits, and the blastp comparison and evolutionary tree analysis know that two The sequences have high similarity with 1Dy10 and 1Dy12 subunits,.KX452081 and KX452082 are mainly composed of signal peptide, N- terminal conservative region, intermediate repeat area (the region is glutamine (Q) and proline (P) enrichment region), C- terminal conserved region I (containing 5 Cys residues), C- end preservation zone II (the region contains rich glutamine (Q) and 1 Cys residues). And the six parts of the conservative region of C- (including 1 Cys residues), through sequence alignment, the composition of N/C terminal amino acids (METRCIPG... VGTGVGAY), the location of the Cys residues and the analysis of the evolutionary tree, found that the two LMW-GS sequences are Glu-D3 loci, Type V (Group 10), M type, and mainly consist of 6 parts: Signal peptide, N- terminal repeat area, polyglutamine region I, characteristic region I, polyglutamine region II and characteristic region II, KX452085 contains 6 Cys residues, which can form 3 pairs of intramolecular two sulfur bonds; KX452086 contains 7 Cys residues, not only can form 3 pairs of intramolecular sulfur bonds, but a superfluous Cys residue may form an intermolecular two sulfur bond. 3. according to the purpose of the study and the results of previous studies, three sequences (KX452083, KX452081 and KX452085) obtained from "Xi Nong 538" were used as the research object. After IPTG induction, they were expressed in the host bacteria Escherichia coli Trans B (DE3) in vitro. The results were identified by SDS-PAGE gel electrophoresis and Western blot. The results showed that all the three proteins were successfully expressed by.4. and three proteins were expressed in vitro. A series of related parameters were obtained by recovery and purification, cryopreservation drying and micronutrient experiments (Chinese Spring flour based flour). These parameters showed that KX452083 (HMW-GS) was positively related to wheat flour, but the elasticity of dough was produced. Negative effects and disadvantageous to the extension of dough; KX452081 (LMW-GS) is more complex and difficult to determine the effect of flour on flour, but it has an enhanced effect on flour elasticity, but the extension of dough is unfavorable. KX452085 (a -gliadin) has a negative effect on the flour flour, but it makes the dough more easy to flow, and the extension and elasticity of the dough have a significant change. In addition, the Farinograph quality number (FQN) is a common index used to evaluate the quality of flour. The quality effect of these three proteins is analyzed and compared with FQN as a measure. The result is: HMW-GS alpha -gliadinLMW-GS.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.1

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