【作者】 王丹；

【导师】 王丕武；

【作者基本信息】 吉林农业大学 ， 作物生物技术， 2012， 硕士

【摘要】 大豆是人们主要的植物蛋白来源,但是在大豆中含有一些抗营养因子如胰蛋白酶抑制剂(KTi)和脂肪氧化酶(Lox)等限制了大豆蛋白质的营养价值和食品风味,在加工过程中,需要通过加热方法去除其活性,这既消耗能量,又降低蛋白的利用率。因此,培育缺失Lox和KTi的大豆新品种,可有效提升大豆的营养价值和加工品质。目前,利用常规育种方法培育缺失KTi和Lox的大豆新品种,受到种质资源有限和育种周期长的限制。利用RNA干扰(RNAi)技术对作物品质进行改良已受到人们的青睐。本研究应用RNA干扰原理构建同时抑制胰蛋白酶抑制剂和脂肪氧化酶基因表达并以除草剂为筛选标记的双价RNAi植物表达载体,利用农杆菌介导法将其导入到大豆品种“吉农28”中,以期下调胰蛋白酶抑制剂基因和脂肪氧化酶基因的表达,得到KTi和Lox含量都降低的转基因大豆新材料。取得如下结果：(1)以单价大豆胰蛋白酶抑制剂KTi基因的RNAi表达载体p7aP-GUS-KTiS的质粒为模板,利用种子特异性启动子的上游引物和NOX终止子的下游引物PCR扩增大豆胰蛋白抑制剂KTi基因完整的RNA干扰构件,插入到单价大豆脂肪氧化酶Lox基因的RNAi植物表达载体pCAMBIA1301-LoxRi中,构建同时含有大豆胰蛋白酶抑制剂KTi基因和脂肪氧化酶Lox基因的无抗生素筛选标记的双价RNAi表达载体,通过质粒PCR和酶切鉴定以及测序结果表明,双价RNAi植物表达载体pCAMBIA1301-KtiRi-LoxRi构建成功。(2)以植物表达载体pCAMBIA3301的质粒DNA为模板,利用CaMV35S启动子的上游引物和CaMV35S终止子的下游引物PCR扩增含有除草剂抗性基因bar的整个表达元件,然后将其插入pCAMBIA1301-KtiRi-LoxRi中,构建以除草剂为筛选标记的双价RNAi表达载体,通过质粒PCR和酶切鉴定以及测序结果表明,双价RNAi植物表达载体pCAMBIA1301-KtiRi-LoxRi-bar构建成功。(3)采用冻融法将双价RNAi植物表载体pCAMBIA1301-KtiRi-LoxRi-bar转入根癌农杆菌EHA101感受态细胞中,经菌落PCR鉴定正确后侵染大豆品种“吉农28”的子叶节,通过除草剂筛选获得再生植株,对转基因植株进行PCR和Southern杂交检测结果表明,成功获得了转基因阳性植株,同时bar基因以单拷贝整合到大豆基因组内。对T1代和T2代转基因植株的PCR检测结果表明,外源基因在转基因T1代和T2代植株中能够遗传。采用RT-PCR测定转基因大豆籽粒中胰蛋白酶抑制剂和脂肪氧化酶基因mRNA的变化,结果表明,在内参基因表达量基本相同的情况下,转基因株系中脂肪氧化酶和胰蛋白酶抑制剂mRNA积累受到明显抑制。以上结果说明双价RNAi表达载体的RNAi表达构件在转基因植株中能正常转录,并分别导致内源胰蛋白酶抑制剂和脂肪氧化酶基因的mRNA在大豆中发生降解。(4)本实验共获得T0代转基因阳性植株10株,T1代转基因阳性植株26株,T2代转基因阳性植株54株。更多还原

【Abstract】 Soybean is the main source of plant protein,but there are some inhibitive factors of nutrition that. which including Kunitz trypsin inhibitors (KTi) and Lipoxygenase. Which Limiting the nutritional value of soy protein and its food flavor. During the manufacturing process, we must through the heating method to remove its activeness. But, heating will decrease the nutrional values of soybeans.Therefore, cultivate new varieties of soybean Which missing lox and kti, and can improve the quality of soybean effectively.We also make good use of the hybrid and backcross breeding method to improve KTti and Lipoxygenase. But, the method can cost a long time for breeding and the germplasm is limited, so they have not reach the purpose of improving the quality of soybean. RNA Interference technique(RNAi) is the main method of transgenic breeding method, Which geting the favour of people. The objective of this study was using RNAi technology to build the plant expression vector Which inhibiting the expression of trypsin inhibitor and lipoxygenase and containing the bar gene. We use Agrobacterium-mediated to import it into soybean varieties of Jinong28, and lowered trypsin inhibitor gene and lipoxygenase. We expect a higher nutritional value of soybean varieties. The main results are listed as following:(1) In this study,we use pCAMBIA1301p7aP-GUS-KTiS as a template used seed-specific promoter upstream primer and the nox terminator down stream primer to clone KTi to insert into pCAMBIA1301-LoxRi, to Construct RNAi Plant Expression Vector of the Lipoxygenase and Kunitz Tripsin Inhibitor which No antibiotic selection marker and named pCAMBIA1301-KtiRi-LoxRi. And Identification of PCR and restriction, and its sequencing.(2) We use pCAMBIA3301as a template to clone bar from it,which using CaMV35S promoter upstream primer and camv35s terminator downstream primer and into pCAMBIA1301-KtiRi-LoxRi and named pCAMBIA1301-KtiRi-LoxRi-bar.And Identification of PCR and restriction, and its sequencing.(3)The ihpRNAseed-specific express edvector pCAMBIA1301-KtiRi-LoxRi-bar were introduced into EHA101respectively. After identification by PCR, the transcojugant was subsequently used to transform soybean cotyledonary node. The transformed explants were screened from barstar.These PCR and Southern blot analysis results of transgenic plant confirmed that the T-DNA region of the ihpRNA seed-specific expressed vector presented in the transgenic plants and integrated into the genome of soybean. hat dsRNA-expressing constructs were inherited in a Mendelian fashion. RT-PCR analysis was performed to detect the levels of endogenous Kti and Lox gene transcripts both in the developing embryos and leaves from the transgenic plants and non-transgenic plant. The soybean tefS1(elongation factor EF-1a) housekeeping gene was used as the internal control; both developing embryos and leaves presented the same level of the tefS1gene. The non-transgenic plant showed Kti and Lox gene expression both in developing embryos, while for the transgenic lines, Kti and Lox gene transcripts were obviously suppressed in the developing embryos. These results of RT-PCR analyses revealed that the accumulation of Kti and Lox mRNA was significantly reduced in the transgenic soybean seeds, and Kti and Lox gene transcripts were obviously suppressed cooperatively in the seeds of transgenic plants with pCAMBIA1301-KtiRi-LoxRi-bar.(4) In this study, we obtained10positive transgenic plants of To,26positive transgenic plants of T1,54positive transgenic plants of T2.更多还原