转hevein和ascorbate peroxidase双基因剑麻对烟草疫霉抗性的研究

发布时间：2018-06-08 22:46

本文选题：剑麻 + APX　；参考：《海南大学》2017年硕士论文

【摘要】：剑麻是世界最重要的麻类经济作物,其应用广泛,综合价值高,前景广阔,但是其受到斑马纹病的危害。剑麻斑马纹病是影响剑麻生产种植的最严重的病害之一,该病致病菌为烟草疫霉菌,是一种传染性病害,会导致剑麻麻区植株染病,最终导致剑麻产量大量降低,严重地制约剑麻产业的可持续发展,造成巨大的经济损失。本研究以提高剑麻品种H.11648抗病性为目的,开展以下研究:1.以pCAMBIA1300为基础载体,构建了病原菌诱导型启动子Hrs203J启动APX 基因和 hevein-like 的双价表达载体:pCAMBIA1300-Hr203J-Hevein-NOS-Hr203J-APX-NOS,用冻融法将载体导入根癌农杆菌EHA105中。2.将从广东广西采集的14株剑麻斑马纹病病原菌进行rDNA-ITS序列分析,结果证明它们都是烟草疫霉菌,并且发现其基因类群与区域有一定联系,通过14株病菌致病力的分析,发现其致病力与菌株所在区域没有直接联系,筛选出致病力较强的2株烟草疫霉菌菌株。3.将植物表达载体 pCAMBIA1300-Hr203J-Hevein-NOS-Hr203J-APX-NOS通过农杆菌介导法对122个剑麻愈伤组织进行遗传转化,获得9株剑麻抗性植株,转化率约为7.4%。对获得的抗性剑麻幼苗进行PCR、RT-PCR检测,证明4株剑麻苗呈阳性,表明APX基因和hevein-like成功导入到剑麻基因组中,阳性转化率为44.4%。并将2株较强的菌株分别接种到4株阳性转基因苗叶片上,其结果为2号转基因剑麻苗株的抗病为2级高抗病,编号1、3、4剑麻苗抗病性呈中抗为3级抗病性。
[Abstract]:Sisal is the most important economic crop of hemp in the world. It is widely used with high comprehensive value and broad prospect, but it is damaged by zebra stripe disease. Sisal zebra stripe is one of the most serious diseases affecting the production and planting of sisal. The pathogen of the disease is Phytophthora tabacum, which is an infectious disease. Seriously restrict the sustainable development of sisal industry, resulting in huge economic losses. In order to improve the disease resistance of sisal variety H. 11648, the following research was carried out: 1. Using pCAMBIA1300 as the basic vector, the bivalent expression vector: pCAMBIA1300-Hr203J-Hevein-NOS-Hr203J-APX-NOSwas constructed, and the vector was transfected into Agrobacterium tumefaciens EHA105 by freeze-thaw method. The bivalent expression vector: pCAMBIA1300-Hr203J-Hevein-Hr203J-APX-NOSwas constructed. The rDNA-ITS sequence analysis of 14 strains of sisal zebra stripe disease collected from Guangdong and Guangxi was carried out. The results showed that they were all Phytophthora tabacum, and found that their gene groups were related to the region, and the pathogenicity of 14 strains were analyzed. It was found that the pathogenicity was not directly related to the region where the strain was located, and two strains of Phytophthora tabacum with strong pathogenicity were screened out. The plant expression vector pCAMBIA1300-Hr203J-Hevein-NOS-Hr203J-APX-NOS was transformed into 122 calli of sisal by Agrobacterium tumefaciens. Nine sisal resistant plants were obtained and the transformation rate was about 7.4%. The results of RT-PCR showed that four sisal seedlings were positive, which indicated that APX gene and hevein-like were successfully introduced into the genome of sisal, and the positive transformation rate was 44.4%. The two stronger strains were inoculated into the leaves of 4 positive transgenic seedlings respectively. The results showed that the disease resistance of transgenic sisal seedling No. 2 was high in grade 2, and the disease resistance of No.2 transgenic sisal seedling was middle to grade 3.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.63

【相似文献】