大豆β-伴大豆球蛋白基因RNAi表达调控机理研究

发布时间：2018-03-27 20:21

本文选题：β-伴大豆球蛋白　切入点：RNAi　出处：《吉林农业大学》2016年博士论文

【摘要】：β-伴大豆球蛋白是大豆蛋白中的主要抗原蛋白,它能引起人和畜禽产生过敏反应,限制了大豆及其产品的广泛应用。因而,有必要降低和消除大豆中β-伴大豆球蛋白的含量,提高其营养价值。目前常用的消除方法有物理方法、化学方法、生物方法等,但这些消除方法效果都不够理想。利用育种方法选育致敏蛋白缺失型大豆是解决大豆抗原蛋白致敏性的最有效方法。传统育种方法受育种周期、种质资源等条件的限制,育成的β-伴大豆球蛋白缺失材料十分有限。近年来迅猛发展起来的RNAi技术为大豆的品质性状改良提供了一条新途径。目前利用RNAi技术对单一基因表达调控的研究较多并已取得成功,但对多基因干扰后表达调控效果的研究尚无报道。β-伴大豆球蛋白主要由α′亚基、α亚基、β亚基三个亚基组成。因此根据RNAi的原理,抑制各亚基基因mRNA的表达量,就能够有效降低β-伴大豆球蛋白在大豆中的含量,从根本上消除其过敏性,进而提高其营养价值。本研究利用RNAi技术分别调控β-伴大豆球蛋白的α′亚基基因、α亚基基因、β亚基基因及α′亚基和β亚基双价基因的表达,利用PCR、实时荧光定量PCR、β-伴大豆球蛋白酶联免疫技术,结合品质分析技术和农艺性状的鉴定,研究和评价RNAi技术对β-伴大豆球蛋白的三个主要亚基基因的表达调控机理和效率,探讨对多个目标基因协同表达调控的新途径,并创制大豆新种质资源。得到下列主要研究结果:1.成功克隆了ihpRNA的两个功能片断,构建了以BADH基因为筛选标记的安全型β-伴大豆球蛋白α亚基基因RNAi表达载体。将该表达载体转化吉农27大豆,经PCR检测,获得了11株T0代阳性植株,4株T1代阳性植株,6株T_2代阳性植株;T_2代转化植株经Southern杂交检测有3株出现杂交信号,表明α亚基基因RNAi表达载体已经以1-2个拷贝形式整合到大豆基因组中;对这3株植株进行实时荧光定量PCR检测,结果表明α亚基基因mRNA表达量明显被抑制,其干扰效率分别为82.3%、85.1%、70.6%;酶联免疫法检测其籽粒中β-伴大豆球蛋白含量,结果表明该蛋白降低了46.42%~63.07%。2.将实验室保存的β-伴大豆球蛋白β亚基基因RNAi表达载体转化吉农27大豆,经PCR检测获得了10株T0代阳性植株,13株T1代阳性植株,6株T_2代阳性植株;T_2代阳性植株Southern杂交显示有2株出现杂交信号,均为单拷贝,表明β-伴大豆球蛋白β亚基基因RNAi表达载体已经以单拷贝形式整合到大豆基因组中;对这2株转基因植株实时荧光定量PCR检测,结果表明β亚基基因的mRNA表达量均明显受到抑制,干扰效率分别为77.5%和82.8%;酶联免疫法检测其籽粒中β-伴大豆球蛋白含量,结果表明该蛋白降低了48.11%~58.73%。3.对实验室保存的转β-伴大豆球蛋白α′亚基RNAi表达载体的T1代PCR阳性大豆种子扩繁,将获得的6株T_2代阳性植株按株行种植,PCR检测结果表明T3、T_4代均有阳性植株检出;T_4阳性植株Southern杂交检测显示有5株出现杂交信号,表明β-伴大豆球蛋白α′亚基基因RNAi表达载体已经以1-2个拷贝形式整合到大豆基因组中;对这5株T_4转基因植株实时荧光定量PCR检测表明α′亚基基因mRNA表达量明显受抑制,干扰效率为43.56%~88.6%;酶联免疫法检测其籽粒中β-伴大豆球蛋白含量,结果表明该蛋白降低了22.67%~69.57%。4.成功构建了β-伴大豆球蛋白α′亚基和β亚基基因双价RNAi表达载体。并将其转入吉农28大豆。经PCR检测获得了11株T0代阳性植株,11株T1代阳性植株,4株T_2代阳性植株;T_2代阳性大豆植株Southern杂交检测显示有3株出现杂交信号,表明构建的双价RNAi表达载体已经以1-2个拷贝形式整合进转基因大豆基因组;对这3株T_2代转基因大豆植株实时荧光定量PCR检测,结果表明α′亚基基因和β亚基基因mRNA表达量均受到明显抑制,其中β亚基基因干扰效率分别为76.80%、86.1%、78.4%,α′亚基基因干扰效率分别为63.2%、62.19%、74.6%;酶联免疫法检测其籽粒中β-伴大豆球蛋白含量,结果表明该蛋白含量降低了46.8%~66.09%。5.利用近红外谷物分析仪,对5株转β-伴大豆球蛋白α′亚基基因RNAi表达载体T_4转基因大豆,测定其蛋白及脂肪含量,结果表明蛋白降低了0.35%~1.47%,脂肪含量提高了0.33%~1.52%,室内考种结果表明这5株T_4转基因大豆植株的主要农艺性状均未发生改变;分别对转β-伴大豆球蛋白α亚基基因RNAi表达载体大豆、转β-伴大豆球蛋白β亚基基因RNAi表达载体大豆及转α′亚基基因和β亚基基因双价RNAi表达载体大豆进行室内考种,结果表明这些T_2代转基因大豆植株的主要农艺性状均未发生改变。
[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.

【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.1

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