华东葡萄VpPR10.1基因转化无核白葡萄及其对霜霉病抗性的研究

发布时间：2018-09-09 17:29

【摘要】：葡萄是世界重要的果树资源,目前全世界栽培最广泛的葡萄多属于欧亚种群。这些栽培品种虽然品质优良,但是抗病性差,给葡萄生产带来了困扰。中国作为葡萄起源地之一,有众多在抗逆方面价值巨大的种质资源,为葡萄分子育种提供了广阔的基因资源。本研究从华东葡萄‘白河35-1’白粉菌诱导cDNA文库中克隆得到VpPR10.1基因,构建植物表达载体,以欧洲葡萄无核白胚性愈伤组织为受体进行遗传转化。在葡萄遗传转化过程中,使用外源多胺以提高转化效率,获得转基因无核白葡萄材料。主要取得以下结果:1.分别使用腐胺、精胺、亚精胺的不同浓度(10μM、100μM和1000μM)在遗传转化的不同时期(共培养阶段、延迟筛选阶段和筛选阶段)对受体材料进行处理。对于添加多胺处理的时期来说,在共培养阶段添加效果优于在延迟筛选阶段添加,优于筛选阶段添加。对于不同种类浓度而言,腐胺1000μM和亚精胺100μM处理效果较好。研究结果表明:外源多胺优化无核白体细胞胚遗传转化体系的最佳处理为:在共培养阶段,培养基为1/2MS培养基+20g/L蔗糖+150μM乙酰丁香酮(AS)另添加1000μM腐胺或100μM亚精胺有助于提高抗性胚在筛选阶段中的诱导率。诱导率分别达到了96.2%和88.8%,而对照组诱导率为44.4%。2.通过农杆菌介导的转化方法得到华东葡萄基因VpPR10.1过表达无核白植株70个,经基因组PCR鉴定和蛋白表达水平鉴定出11个转基因植株,阳性率为15.7%。3.使用VpPR10.1过表达转基因株系6904和野生型无核白离体叶片接种霜霉菌,苯胺蓝染色结果显示转基因株系6904上霜霉菌生长较野生型受到抑制,在接种后1-5天的观察期内,接菌后2天菌丝上的吸器数量显著减少;接种5天后统计单位面积产孢量,6904株系离体叶盘上的产孢量也显著降低;DAB染色结果显示,接种后76h、96h转基因株系6904离体叶盘上细胞内活性氧积累较野生型明显,有明显的超敏反应。转华东葡萄VpPR10.1基因能够提高无核白葡萄对霜霉病的抗性。
[Abstract]:Grape is an important fruit resource in the world. At present, most of the most widely cultivated grapes in the world belong to Eurasian population. Although the quality of these cultivars is good, but the disease resistance is poor, which brings trouble to grape production. As one of the places of grape origin, China has many germplasm resources of great value in stress resistance, which provides broad genetic resources for grape molecular breeding. In this study, the VpPR10.1 gene was cloned from cDNA library of 'Baihe 35-1' Grape in East China, and the plant expression vector was constructed, and the non-white embryogenic callus of European grape was used as the receptor for genetic transformation. In the process of grape genetic transformation, exogenous polyamines were used to improve the efficiency of transformation and to obtain transgenic non-white grape materials. The main results are as follows: 1. The receptor materials were treated with putrescine, spermine and spermidine (10 渭 m 100 渭 M and 1000 渭 M) at different stages of genetic transformation (co-culture stage, delayed screening stage and screening stage) respectively. For the period of adding polyamines, the effect of adding polyamines in co-culture stage is better than that in delayed screening stage and in screening stage. For different kinds of concentration, putrescine 1000 渭 M and spermidine 100 渭 M had better effect. The results showed that the optimal treatment of exogenous polyamines to optimize the genetic transformation system of non-nuclear white somatic embryos was as follows: in co-culture stage, The addition of 1000 渭 M putrescine or 100 渭 M spermidine to the 1/2MS medium 20g/L sucrose 150 渭 M acetyl eugenone (AS) could increase the induction rate of resistant embryos in the screening stage. The induction rate was 96.2% and 88.8%, respectively, while that of the control group was 44.4% and 88.8%, respectively. 70 transgenic plants were obtained by Agrobacterium tumefaciens mediated transformation. Eleven transgenic plants were identified by genomic PCR identification and protein expression level, and the positive rate was 15.70.3. VpPR10.1 overexpression transgenic lines 6904 and wild type denuclearized leaves were inoculated in vitro. The results of aniline blue staining showed that the growth of the transgenic strain 6904 was more inhibited than that of wild type, and during the observation period of 1-5 days after inoculation. 2 days after inoculation, the number of hyphal absorbers was significantly reduced, and the spore production per unit area was counted after 5 days inoculation, and the spore production on leaves of isolated leaves of strain 6904 was also significantly decreased by DAB staining. The accumulation of reactive oxygen species (Ros) in the leaves of the transgenic strain 6904 at 76 h and 96 h after inoculation was significantly higher than that of the wild type and showed obvious hypersensitivity. Transgenic grape VpPR10.1 gene can improve the resistance to downy mildew in non-nuclear white grape.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.631

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