农杆菌介导的抗草甘膦基因对玉米自交系18-599幼胚的遗传转化

发布时间：2018-09-07 09:05

【摘要】：玉米作为三大粮食作物之一,在饲料生产中扮演重要的角色,也为生物能源制造提供良好原材料；伴随人口的增长、人民生活水平的提高,以及畜牧业的极速发展,对玉米需求量显著增加。田间的杂草与玉米竞争阳光和肥料,形成草害,引起减产,严重制约了玉米产量的增加。人工拨草和机械除草费时费力且效果不理想；化学除草方便、经济有效,业已成为现代农业技术重要组成部分。目前,全世界农业生产中使用量最大最广泛的除草剂是草甘膦。由于草甘膦杀草谱比较宽,对玉米也具有灭生性作用,这就使在玉米生产中使用草甘膦受到限制。因此,培育出具有自主知识产权的抗草甘膦的玉米新品种,进行试验、示范、推广,能降低除草成本和劳动强度、提高工作效率、增强我国玉米的国际市场竞争力。本研究主要是将抗草甘膦的抗性基因2mg2-epsps插入到植物表达载体pCAMBIA3301中,以农杆菌菌株EHA105携带含有目的基因的表达载体进行介导转化,遗传转化受体材料为自交系18-599R的幼胚；实验首先探讨了热激与离心对幼胚浸染的影响,并对农杆菌转化玉米幼胚过程中幼胚大小、筛选剂浓度,头孢霉素浓度等因素进行优化,为建立良好的玉米幼胚转化体系奠定基础；获得抗草甘膦转基因玉米植株,并进行的PCR检测与产物测序,结果表明目的基因已整合到玉米基因组中；对阳性植株进行叶面喷施草甘膦,结果表明转基因玉米后代具有草甘膦抗性。主要研究结果如下： 1目的基因的获得。以表达载体P35S-2301-EPSPS为模板,用高保真酶扩增获得目的基因片段并测序,利用NCBI的在线软件Blast、ORF Finder分析,结果表明,扩增的序列全长为1563bp,编码464个氨基酸；与原始载体基因碱基序列同源性为99%,氨基酸序列匹配度为100%。 2表达载体构建。将构建好pMD19T-2mg2-epsps载体和pCAMBIA3301表达载体同时进行双酶切,回收目的片段并进行连接转化；经酶切、测序验证结果表明所连接确实为目的片段,没有发生突变。 3玉米幼胚遗传转化系统的优化。首先探讨了热激与离心对幼胚浸染的影响,并对农杆菌转化玉米幼胚过程中幼胚大小、头孢霉素浓度、筛选剂浓度等因素设置不同水平处理,结果表明：最适合的处理条件为热激40℃,3min,冷处理25℃,2min,不离心,此处理得到最高的抗性愈伤得率为52%；浸染的幼胚大小为0.8-1.3mm时,可以得到质地较好的愈伤组织；头孢霉素为400mg/L的浓度的不仅可以抑制农杆菌生长,还可以保持幼胚良好的生长状态；利用不同浓度的草甘膦对浸染的幼胚进行筛选,比较理想的筛选浓度为1.5-2.0mmol/L。 4转基因苗的PCR鉴定。对96株To代再生植株进行多次重复的PCR鉴定,15株再生植株的基因组DNA扩增出与阳性对照大小一致的的片段,表明目的基因已经成功转入玉米基因组中；T1代株系进行PCR检测,获得四株转基因株系,这些阳性的株系经RT-PCR检测,表明抗草甘膦基因在mRNA水平上得到了有效转录；对阳性植株进行叶面喷施草甘膦,结果表明转基因玉米后代具有草甘膦抗性。。
[Abstract]:As one of the three major grain crops, maize plays an important role in feed production and provides good raw materials for bioenergy production. With the growth of population, the improvement of people's living standards and the rapid development of animal husbandry, the demand for maize has increased significantly. At present, glyphosate is the most widely used herbicide in agricultural production in the world. Because of the comparison of glyphosate herbicidal spectrum, it has become an important part of modern agricultural technology. Wide, also has a sterile effect on corn, which limits the use of glyphosate in Maize production. Therefore, cultivating new varieties of corn with independent intellectual property rights of glyphosate resistance, testing, demonstration, promotion, can reduce the cost of weeding and labor intensity, improve work efficiency, enhance the competitiveness of China's Maize in the international market.
In this study, the glyphosate-resistant gene 2mg 2-epsps was inserted into the plant expression vector pCAMBIA 3301. Agrobacterium strain EHA105 carrying the expression vector containing the target gene was used to mediate the transformation, and the genetic transformation receptor material was the immature embryo of inbred line 18-599R. The size of immature embryo, the concentration of screening agent and cephalosporin in the process of transforming maize immature embryo by Agrobacterium tumefaciens were optimized to lay a foundation for establishing a good system of transforming maize immature embryo. Glyphosate was sprayed on the leaves of the positive plants. The results showed that the progeny of transgenic maize had glyphosate resistance.
1. The target gene was obtained by using P35S-2301-EPSPS as the template and amplified by high-fidelity enzymes. The sequence was analyzed by NCBI on-line software Blast and ORF Finder. The results showed that the amplified sequence was 1 563 BP in length and encoded 464 amino acids. The homology with the base sequence of the original vector gene was 99%, and the amino acid sequence was 99%. The matching degree is 100%.
The pMD19T-2mg 2-epsps vector and the pCAMBIA3301 expression vector were digested by double enzyme digestion, and the target fragment was recovered and transformed. The results of enzyme digestion and sequencing showed that the fragment was indeed the target fragment, and no mutation occurred.
3. Optimization of genetic transformation system of immature embryo of maize. Firstly, the effect of heat shock and centrifugation on immature embryo dissemination was discussed, and different levels of treatment were set for immature embryo size, cephalosporin concentration, screening agent concentration and other factors in the process of transformation of immature embryo of Maize by Agrobacterium tumefaciens. The results showed that the most suitable treatment conditions were heat shock 40 C, 3 min, cold treatment 25 C Without centrifugation, the highest yield of resistant callus was 52%; when the immature embryos were 0.8-1.3 mm in size, the callus with better texture could be obtained; the concentration of cephalosporin 400 mg/L could not only inhibit the growth of Agrobacterium tumefaciens, but also keep the immature embryos in good growth state; different concentrations of glyphosate could be used to immerse the immature embryos. The embryos were screened with an ideal screening concentration of 1.5-2.0mmol/L..
4. PCR identification of transgenic seedlings. Repeated PCR identification of 96 Togeneration regenerated plants showed that the genomic DNA of 15 regenerated plants produced fragments of the same size as the positive control, indicating that the target gene had been successfully transferred into the maize genome. Four transgenic lines were obtained from T1 generation by PCR detection. RT-PCR showed that glyphosate-resistant genes were effectively transcribed at the mRNA level, and glyphosate was sprayed on the leaves of the positive plants. The results showed that the progenies of transgenic maize had glyphosate resistance.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S513

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