本文選題：普通小麥 + 高分子量谷蛋白　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：小麥貯藏蛋白分兩種:麥谷蛋白(Glutenin)和醇溶蛋白(Gliadin),麥谷蛋白也分兩種:高分子量谷蛋白亞基(high molecular weight glutenin subunit,HMW-GS)和低分子量谷蛋白亞基(low molecular weight glutenin subunit,LMW-GS)。小麥貯藏蛋白是小麥面粉加工品質(zhì)的決定性因素,其含量超過(guò)小麥籽粒蛋白總量的80%。本研究以課題組自主選育的優(yōu)質(zhì)小麥新品種“西農(nóng)538”和“西農(nóng)558”的基因組DNA為模板,根據(jù)GenBank中收錄的HMW-GS、LMW-GS和α-gliadin的基因序列,設(shè)計(jì)特異性引物,通過(guò)PCR擴(kuò)增、克隆測(cè)序等實(shí)驗(yàn)技術(shù),分別從這兩個(gè)小麥品種中得到了三種蛋白的基因編碼序列各一條,并進(jìn)一步進(jìn)行了序列比對(duì)及進(jìn)化樹(shù)分析,同時(shí)以“西農(nóng)538”中獲得的三條基因序列為目標(biāo)序列,誘導(dǎo)其在大腸桿菌表達(dá)系統(tǒng)中表達(dá),然后通過(guò)蛋白純化和微量摻粉實(shí)驗(yàn)研究比較這三個(gè)表達(dá)蛋白各自對(duì)小麥面粉的品質(zhì)效應(yīng)。實(shí)驗(yàn)結(jié)果如下:1.利用設(shè)計(jì)的特異性引物,共得到了6條具有單一完整開(kāi)放閱讀框的基因序列。其中從“西農(nóng)538”中克隆得到的HMW-GS、LMW-GS和α-gliadin基因序列的GenBank登錄號(hào)分別為KX452083、KX452081、KX452085,編碼區(qū)長(zhǎng)度分別為1812 bp、915 bp和897 bp;從“西農(nóng)558”中克隆得到的這三種蛋白的基因序列的登錄號(hào)分別為KX452084、KX452082、KX452086,編碼區(qū)長(zhǎng)度分別為1977 bp、915 bp和879 bp。2.通過(guò)氨基酸序列對(duì)比發(fā)現(xiàn):KX452083和KX452084主要由信號(hào)肽、N-端保守區(qū)、中間重復(fù)區(qū)和C-末端四個(gè)部分構(gòu)成,都有7個(gè)半胱氨酸(Cys)殘基,中間重復(fù)區(qū)有六肽和九肽重復(fù)單元,但沒(méi)有三肽重復(fù)單元,由此推測(cè)這兩個(gè)基因序列可能屬于y型亞基,同時(shí)經(jīng)過(guò)blastp比對(duì)和進(jìn)化樹(shù)分析得知,兩條序列分別與1Dy10和1Dy12亞基有極高的相似度。KX452081和KX452082主要由信號(hào)肽、N-端保守區(qū)、中間重復(fù)區(qū)(該區(qū)域是谷氨酰胺(Q)和脯氨酸(P)的富集區(qū))、C-端保守區(qū)Ⅰ(含有5個(gè)Cys殘基)、C-端保守區(qū)Ⅱ(該區(qū)域含有豐富的谷氨酰胺(Q)和1個(gè)Cys殘基)和C-端保守區(qū)Ⅲ(含有1個(gè)Cys殘基)這六部分組成,通過(guò)序列比對(duì)、N/C端氨基酸組成(METRCIPG······VGTGVGAY)、Cys殘基的位置和進(jìn)化樹(shù)分析發(fā)現(xiàn),得到的這兩條LMW-GS序列屬于Glu-D3位點(diǎn)、Type V(Group 10)、m型、C組LMW-GS。KX452085和KX452086主要由6部分組成:信號(hào)肽、N-端重復(fù)區(qū)、多聚谷氨酰胺區(qū)Ⅰ、特征區(qū)Ⅰ、多聚谷氨酰胺區(qū)Ⅱ和特征區(qū)Ⅱ,其中KX452085含有6個(gè)Cys殘基,可形成3對(duì)分子內(nèi)二硫鍵;KX452086含有7個(gè)Cys殘基,不僅可以形成3對(duì)分子內(nèi)二硫鍵,多余的一個(gè)Cys殘基可能形成一個(gè)分子間二硫鍵。3.依據(jù)本實(shí)驗(yàn)的研究目的和前人的研究成果,以“西農(nóng)538”中克隆得到的三條序列(KX452083、KX452081和KX452085)為研究對(duì)象,經(jīng)IPTG誘導(dǎo),使它們?cè)谒拗骶鶨scherichia coli Trans B(DE3)中進(jìn)行體外表達(dá),通過(guò)SDS-PAGE凝膠電泳和Western blot檢測(cè)鑒定,結(jié)果表明這三個(gè)蛋白均成功表達(dá)。4.對(duì)三個(gè)蛋白進(jìn)行大量的體外表達(dá),經(jīng)回收純化、低溫冷凍干燥和微量摻粉實(shí)驗(yàn)(以中國(guó)春面粉為基礎(chǔ)面粉),得到了一系列相關(guān)參數(shù)。分析這些參數(shù)發(fā)現(xiàn),KX452083(HMW-GS)與面粉筋力正相關(guān),但對(duì)面團(tuán)彈性產(chǎn)生了負(fù)效應(yīng),并且不利于面團(tuán)的延伸;KX452081(LMW-GS)對(duì)面粉筋力的影響比較復(fù)雜,尚難確定相關(guān)性,但其對(duì)面粉彈性有增強(qiáng)作用,而對(duì)面團(tuán)的延伸不利;KX452085(α-gliadin)對(duì)面粉筋力有負(fù)效應(yīng),但使面團(tuán)更易流變,且對(duì)面團(tuán)的延伸和彈性有顯著的改善。此外,粉質(zhì)質(zhì)量指數(shù)(Farinograph quality number,FQN)是一種被用來(lái)評(píng)價(jià)面粉質(zhì)量的常用指標(biāo),以FQN為衡量標(biāo)準(zhǔn)分析比較這三個(gè)蛋白的品質(zhì)效應(yīng),結(jié)果為: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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S512.1

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