發(fā)布時(shí)間：2018-03-27 20:21

  本文選題：β-伴大豆球蛋白　切入點(diǎn)：RNAi　出處：《吉林農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：β-伴大豆球蛋白是大豆蛋白中的主要抗原蛋白,它能引起人和畜禽產(chǎn)生過敏反應(yīng),限制了大豆及其產(chǎn)品的廣泛應(yīng)用。因而,有必要降低和消除大豆中β-伴大豆球蛋白的含量,提高其營(yíng)養(yǎng)價(jià)值。目前常用的消除方法有物理方法、化學(xué)方法、生物方法等,但這些消除方法效果都不夠理想。利用育種方法選育致敏蛋白缺失型大豆是解決大豆抗原蛋白致敏性的最有效方法。傳統(tǒng)育種方法受育種周期、種質(zhì)資源等條件的限制,育成的β-伴大豆球蛋白缺失材料十分有限。近年來迅猛發(fā)展起來的RNAi技術(shù)為大豆的品質(zhì)性狀改良提供了一條新途徑。目前利用RNAi技術(shù)對(duì)單一基因表達(dá)調(diào)控的研究較多并已取得成功,但對(duì)多基因干擾后表達(dá)調(diào)控效果的研究尚無報(bào)道。β-伴大豆球蛋白主要由α′亞基、α亞基、β亞基三個(gè)亞基組成。因此根據(jù)RNAi的原理,抑制各亞基基因mRNA的表達(dá)量,就能夠有效降低β-伴大豆球蛋白在大豆中的含量,從根本上消除其過敏性,進(jìn)而提高其營(yíng)養(yǎng)價(jià)值。本研究利用RNAi技術(shù)分別調(diào)控β-伴大豆球蛋白的α′亞基基因、α亞基基因、β亞基基因及α′亞基和β亞基雙價(jià)基因的表達(dá),利用PCR、實(shí)時(shí)熒光定量PCR、β-伴大豆球蛋白酶聯(lián)免疫技術(shù),結(jié)合品質(zhì)分析技術(shù)和農(nóng)藝性狀的鑒定,研究和評(píng)價(jià)RNAi技術(shù)對(duì)β-伴大豆球蛋白的三個(gè)主要亞基基因的表達(dá)調(diào)控機(jī)理和效率,探討對(duì)多個(gè)目標(biāo)基因協(xié)同表達(dá)調(diào)控的新途徑,并創(chuàng)制大豆新種質(zhì)資源。得到下列主要研究結(jié)果:1.成功克隆了ihpRNA的兩個(gè)功能片斷,構(gòu)建了以BADH基因?yàn)楹Y選標(biāo)記的安全型β-伴大豆球蛋白α亞基基因RNAi表達(dá)載體。將該表達(dá)載體轉(zhuǎn)化吉農(nóng)27大豆,經(jīng)PCR檢測(cè),獲得了11株T0代陽(yáng)性植株,4株T1代陽(yáng)性植株,6株T_2代陽(yáng)性植株;T_2代轉(zhuǎn)化植株經(jīng)Southern雜交檢測(cè)有3株出現(xiàn)雜交信號(hào),表明α亞基基因RNAi表達(dá)載體已經(jīng)以1-2個(gè)拷貝形式整合到大豆基因組中;對(duì)這3株植株進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè),結(jié)果表明α亞基基因mRNA表達(dá)量明顯被抑制,其干擾效率分別為82.3%、85.1%、70.6%;酶聯(lián)免疫法檢測(cè)其籽粒中β-伴大豆球蛋白含量,結(jié)果表明該蛋白降低了46.42%~63.07%。2.將實(shí)驗(yàn)室保存的β-伴大豆球蛋白β亞基基因RNAi表達(dá)載體轉(zhuǎn)化吉農(nóng)27大豆,經(jīng)PCR檢測(cè)獲得了10株T0代陽(yáng)性植株,13株T1代陽(yáng)性植株,6株T_2代陽(yáng)性植株;T_2代陽(yáng)性植株Southern雜交顯示有2株出現(xiàn)雜交信號(hào),均為單拷貝,表明β-伴大豆球蛋白β亞基基因RNAi表達(dá)載體已經(jīng)以單拷貝形式整合到大豆基因組中;對(duì)這2株轉(zhuǎn)基因植株實(shí)時(shí)熒光定量PCR檢測(cè),結(jié)果表明β亞基基因的mRNA表達(dá)量均明顯受到抑制,干擾效率分別為77.5%和82.8%;酶聯(lián)免疫法檢測(cè)其籽粒中β-伴大豆球蛋白含量,結(jié)果表明該蛋白降低了48.11%~58.73%。3.對(duì)實(shí)驗(yàn)室保存的轉(zhuǎn)β-伴大豆球蛋白α′亞基RNAi表達(dá)載體的T1代PCR陽(yáng)性大豆種子擴(kuò)繁,將獲得的6株T_2代陽(yáng)性植株按株行種植,PCR檢測(cè)結(jié)果表明T3、T_4代均有陽(yáng)性植株檢出;T_4陽(yáng)性植株Southern雜交檢測(cè)顯示有5株出現(xiàn)雜交信號(hào),表明β-伴大豆球蛋白α′亞基基因RNAi表達(dá)載體已經(jīng)以1-2個(gè)拷貝形式整合到大豆基因組中;對(duì)這5株T_4轉(zhuǎn)基因植株實(shí)時(shí)熒光定量PCR檢測(cè)表明α′亞基基因mRNA表達(dá)量明顯受抑制,干擾效率為43.56%~88.6%;酶聯(lián)免疫法檢測(cè)其籽粒中β-伴大豆球蛋白含量,結(jié)果表明該蛋白降低了22.67%~69.57%。4.成功構(gòu)建了β-伴大豆球蛋白α′亞基和β亞基基因雙價(jià)RNAi表達(dá)載體。并將其轉(zhuǎn)入吉農(nóng)28大豆。經(jīng)PCR檢測(cè)獲得了11株T0代陽(yáng)性植株,11株T1代陽(yáng)性植株,4株T_2代陽(yáng)性植株;T_2代陽(yáng)性大豆植株Southern雜交檢測(cè)顯示有3株出現(xiàn)雜交信號(hào),表明構(gòu)建的雙價(jià)RNAi表達(dá)載體已經(jīng)以1-2個(gè)拷貝形式整合進(jìn)轉(zhuǎn)基因大豆基因組;對(duì)這3株T_2代轉(zhuǎn)基因大豆植株實(shí)時(shí)熒光定量PCR檢測(cè),結(jié)果表明α′亞基基因和β亞基基因mRNA表達(dá)量均受到明顯抑制,其中β亞基基因干擾效率分別為76.80%、86.1%、78.4%,α′亞基基因干擾效率分別為63.2%、62.19%、74.6%;酶聯(lián)免疫法檢測(cè)其籽粒中β-伴大豆球蛋白含量,結(jié)果表明該蛋白含量降低了46.8%~66.09%。5.利用近紅外谷物分析儀,對(duì)5株轉(zhuǎn)β-伴大豆球蛋白α′亞基基因RNAi表達(dá)載體T_4轉(zhuǎn)基因大豆,測(cè)定其蛋白及脂肪含量,結(jié)果表明蛋白降低了0.35%~1.47%,脂肪含量提高了0.33%~1.52%,室內(nèi)考種結(jié)果表明這5株T_4轉(zhuǎn)基因大豆植株的主要農(nóng)藝性狀均未發(fā)生改變;分別對(duì)轉(zhuǎn)β-伴大豆球蛋白α亞基基因RNAi表達(dá)載體大豆、轉(zhuǎn)β-伴大豆球蛋白β亞基基因RNAi表達(dá)載體大豆及轉(zhuǎn)α′亞基基因和β亞基基因雙價(jià)RNAi表達(dá)載體大豆進(jìn)行室內(nèi)考種,結(jié)果表明這些T_2代轉(zhuǎn)基因大豆植株的主要農(nóng)藝性狀均未發(fā)生改變。
[Abstract]:Beta conglycinin is the main antigen protein in soybean protein, it can cause allergic reactions in people and livestock, limiting the application of soybean and its products. Therefore, it is necessary to reduce and eliminate the content in soybean beta conglycinin, improve its nutritional value. The method to eliminate common physical method, chemical method, biological method, but these methods are not ideal. To eliminate the effect of using the breeding method of allergenic protein deficient soybean breeding is the most effective method to solve the soybean antigen protein allergenicity. Traditional breeding methods by the period of breeding, germplasm resources and other conditions, the incubation of beta with deletion of soybean materials the globulin is limited. In recent years the rapid development of RNAi technology provides a new way for improving the quality traits of soybean. At present, the use of RNAi technology in single gene expression regulation and has taken more research Well, there is no report on study but gene expression regulation after interference effect. Beta conglycinin is mainly composed of alpha 'subunit, alpha subunit, beta subunit of three subunits. Therefore, according to the principle of RNAi, inhibit the expression of the subunit gene mRNA, can effectively reduce the content of beta conglycinin in soybeans, eliminating the allergic fundamentally and improve its nutritional value.' alpha subunit gene in this study using RNAi technology to regulate beta conglycinin, alpha subunit gene, beta subunit gene and 'alpha and beta subunit double gene expression by PCR, real-time PCR, beta conglycinin enzyme immunoassay, analysis and identification of technical quality and agronomic traits with the mechanism of expression regulation and efficiency of research and evaluation of RNAi technology of three main subunit genes of beta conglycinin, explore the gene of a target Co expression of new ways of regulation, and create new soybean germplasm resources. The following are the main results: 1. successfully cloned two functional fragments of ihpRNA was constructed using BADH gene as a selection marker safety type beta conglycinin alpha subunit gene RNAi expression vector. The expression vector was transformed into Jinong 27 soybean, detected by PCR, obtained 11 strains of T0 transgenic plants, 4 strains of T1 transgenic plants, 6 strains of T_2 transgenic plants; T_2 generation transgenic plants by Southern hybridization detection of 3 strains showed that the hybridization signals appear, alpha subunit gene RNAi expression vector has 1-2 copy form integrated into the soybean genome; real-time fluorescence quantitative PCR detection of the 3 plants, the results showed that the expression of alpha subunit gene mRNA was obviously inhibited, the interference efficiency were 82.3%, 85.1%, 70.6%; enzyme linked immunosorbent assay in the grain - conglycinin content, the results show that the Protein reduced 46.42%~63.07%.2. beta gene RNAi with preservation of laboratory soybean protein beta subunit expression vector into Jinong 27 soybean, were obtained by PCR detection of 10 strains of T0 transgenic plants, 13 strains of T1 transgenic plants, 6 strains of T_2 transgenic plants; T_2 transgenic plants of Southern hybridization showed that 2 isolates appeared hybridization signals were single copy, showed that beta RNAi gene with soybean protein beta subunit expression vector in the form of single copy has been integrated into the soybean genome; the real-time fluorescence quantitative PCR these 2 transgenic plants were detected, suggesting that beta subunit gene mRNA expression was significantly inhibited by the interference efficiency, respectively. 77.5% and 82.8%; enzyme linked immunosorbent assay in the grain beta conglycinin content, the results show that the protein 48.11%~58.73%.3. decreased on beta conglycinin preservation laboratory alpha 'subunit RNAi expression vector T1 PCR Yang Soybean seed propagation, 6 strains of T_2 transgenic plants will be obtained according to plant cultivation, PCR test results show that the T3 and T_4 generation were detected positive plants; detection of T_4 positive plants of Southern hybridization showed that 5 isolates showed hybridization signal, suggesting that the beta conglycinin 'alpha subunit gene RNAi expression vector has to 1-2 copies integrated into the soybean genome; the real-time fluorescence quantitative PCR these 5 strains of T_4 transgenic plants showed that protein kinase mRNA expression was inhibited, the interference efficiency of 43.56%~88.6%; enzyme linked immunosorbent assay in the grain beta conglycinin content, the results show that the protein decreased 22.67%~69.57%.4. the construction of beta with bivalent glycinin' alpha and beta subunit gene RNAi expression vector. And put it into Jinong 28. Soybean were obtained by PCR detection of 11 strains of T0 transgenic plants, 11 strains of T1 transgenic plants, 4 plants of T_2 generation Yang Plant; detection of T_2 positive soybean plants Southern hybridization showed that 3 isolates showed hybridization signal, RNAi showed that the bivalent expression vector constructed by using 1-2 copies of the form has been integrated into the genome of the transgenic soybean; 3 strains of T_2 generation transgenic soybean plant real-time fluorescence quantitative PCR detection results show that protein kinase gene and beta subunit gene expression of mRNA was significantly inhibited, the beta subunit gene interference efficiency were 76.80%, 86.1%, 78.4%, 'alpha subunit gene interference efficiency were 63.2%, 62.19%, 74.6%; enzyme linked immunosorbent assay in the grain beta conglycinin content, the results show that the protein content was reduced by 46.8%~66.09%.5. using near infrared grain analyzer, 5 strains of transgenic beta conglycin' alpha subunit gene RNAi expression vector of T_4 transgenic soybean, determination of protein and fat content. The results show that reduced 0.35%~1.47% protein, fat The content of 0.33%~1.52% increased, the indoor test results showed that the 5 strains of Main Agronomic Traits of T_4 transgenic soybean plants were not changed; respectively of beta conglycinin protein alpha subunit gene expression vector RNAi beta gene with soybean, transgenic soybean RNAi globulin subunit expression vector and transgenic soybean bivalent 'alpha subunit gene and beta subunit gene RNAi expression vector lab test results show that the soybean, the main agronomic traits of T_2 generation transgenic soybean plants were not changed.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S565.1

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