【作者】 张军；

【导师】 李杰；

【作者基本信息】 东北农业大学 ， 遗传学， 2012， 硕士

【摘要】 玉米是世界上重要的粮食作物和饲料作物,但由于玉米中赖氨酸含量偏低,致使玉米中蛋白质的实际利用率只有40%-50%。同时随着人们生活水平的提高、膳食结构的改变,对玉米蛋白品质的要求越来越高。另一方面,由于虫害的发生,玉米产量遭到极大的损失,其中以玉米螟危害最重。通过基因工程方法将高赖氨酸蛋白基因和抗虫基因转入玉米中,从而改善玉米的蛋白品质,降低虫害对玉米产量的影响具有重要的意义。本实验选取7个玉米自交系进行愈伤组织诱导,以诱导出的较好的胚性愈伤组织为受体,利用农杆菌介导法对其进行遗传转化,将分别带有高赖氨酸蛋白基因GsHLP2、抗虫基因crylla8和bar选择标记基因的双T-DNA表达载体转化玉米愈伤组织,筛选共转化植株。为通过转基因植株后代的遗传分离培育无选择标记的转基因玉米提供材料。主要研究结果如下：1.抗虫基因crylla8植物表达载体的构建构建了由带有内含子的组成型强启动子Ubi调控的cry1Ia8基因植物表达载体,用于农杆菌介导法转化。2.农杆菌介导的玉米自交系遗传转化体系的建立对7个玉米自交系和4种培养基组的合进行筛选,选择出了最适合诱导愈伤组织的自交系、培养基组合。其中B73(N6)和HiⅡB(NB)组合的初始愈伤诱导率和胚性愈伤诱导率分别为82.3%、73.9%和70.5%、63.8%,相比之下B73(N6)组合的诱导率和愈伤状态都优于HiⅡB(NB)组合,故选择B73的愈伤组织作为遗传转化受体。对B73玉米胚性愈伤组织农杆菌介导的转化体系进行了优化,确立了较为较优的转化条件：农杆菌菌液浓度为OD600=0.6,侵染时间为20min,共培养温度为23℃。并确定了B73玉米胚性愈伤组织的双丙氨磷筛选压为2mg/L。3.转基因玉米植株的筛选和鉴定用农杆菌介导法对B73玉米自交系胚性愈伤组织进行转化,得到了160株再生植株,经Basta涂抹筛得到41株抗性植株,经检测34株为bar基因PCR阳性。其中,3株高赖氨酸基因PCR阳性,4株crylla8基因PCR阳性。更多还原

【Abstract】 Maize (Zea Maya L.) is an importment cereal crop in the world today, and china is the second largest corn-producing country in the world.However, because the low content of lysine in the maize, the actual utilization of protein in the maize is only40%-50%.With the improvement of people living standards and structure changes in dietary, the demand to improve the puality of people of maize protein is increasing. On the other hand, because of the occurrence of pests, result in a great loss to the maize production, and the Pyrausta nubilalis (Hubern) is the main pest in order to improve the quality of maize and enhance it’s produetion, it’s important to use the genetic engineering method to transfer target gene into maize. It’s applicable to use Agrobacterium-mediated systerm to obtain transgenic plants of maize.In this study, seven corn inbred lines were cultured to induction the callus. Choose the good embryogenic callus as the receptor of Agrobacterium-mediated genetic transformation.And the double T-DNA expression vector that with crylla8and bar or with GsHLP2and bar was used in the transformed of maize callus, Screening the plants of co-transformationed. Provide material for the cultivate of marker-free transgenic maize by genetic segregation of the offspring of transgenic plants.The major results were as follows:1. Construction of plant expression vector of cryⅡa8gene.Constructed the plant expression vectors of crylla8gene that controled by the Constitutively strong Ubi promotor and it’s intron.2. Establish the system of genetic transformation of Agrobacterium-mediated of maize inbred lines.Seven combination of maize inbred lines and media were filted, select the most appropriate combination for callus induction inbred lines and media, the initial callus induction rate of B73(N6)和HiⅡB (NB) is82.3%、73.9%and the embryogenic callus induction rate of B73(N6)和HiⅡB (NB) is70.5%、63.8%, the combination of B73(N6) is better than HiⅡB (NB),so choose the callus of B73as the acceptor of genetic transformation The systerm of tissye culture of maize and transformation has been optimized and established a optimal condition of Agrobacterium-mediated transformation of embrygenic callus of maize inbred line.the optimized condition is:the numerical value of OD600of Agrobacterium is0.6, the time of infect is20min and the temperature of co-culture is23℃. The bialaphos screen pressure of immature maize calli of B73is2mg/L。3. The screening and identification of Transgenic maize plantsThe embryonic calli of B73were transformationed by Agrobacterium-mediated method, obtained160regenerated plants, PCR analysis showed that there were34regenerated plants with the intergration of bar gene, including4plants with the intergration of cry1Ia8gene,3plants with the intergration of GsHLP2gene and1plant fructify in the3plants.更多还原