【作者】 张娜；

【导师】 徐炎； 刘国甜；

【作者基本信息】 西北农林科技大学 ， 果树学， 2021， 硕士

【摘要】 葡萄霜霉病是严重危害葡萄生产的一大卵菌病害。引起霜霉病的葡萄霜霉菌(Plasmopara viticola)是一种专性寄生卵菌,不同葡萄品种/种对霜霉菌的抗性存在着显著差异。欧洲葡萄(Vitis vinifera L.)易感霜霉病,而我国野生葡萄表现出对霜霉病的抗性。近年来WRKY转录因子已被证实参与植物的防御反应,因此挖掘抗病葡萄品种中的抗病基因改良欧洲葡萄对于葡萄育种有着极其重要的意义。实验室前期从中国野生复叶葡萄‘留坝-8’中筛选到了一个受霜霉菌诱导表达的转录因子VpWRKY75以及预测出其可能互作的下游靶基因PR10.8。本研究以此为基础,利用VpWRKY75同源基因AtWRKY75拟南芥突变体和葡萄愈伤组织分析了WRKY75初步的抗病功能以及对植物激素的响应,并通过双荧光素酶和酵母单杂交实验进行了VpWRKY75与VpPR10.8启动子的互作验证。最后通过农杆菌介导的葡萄遗传转化体系进行WRKY75基因的过表达以及敲除,以期获得转基因葡萄植株。取得的主要研究结果如下:(1)对VpWRKY75同源基因AtWRKY75拟南芥突变体进行病原菌接种处理,初步分析WRKY75的抗病功能。接种与霜霉菌同属卵菌的辣椒疫霉菌后,与野生型相比,突变体株系的病斑面积更大,相对生物量更多,细胞死亡也更多;接种Pst DC3000后,突变体拟南芥叶片表现出比野生型更敏感的表型:叶片黄化更严重,细菌数量也显著增多,而细胞程序性死亡与过氧化氢明显较少;接种坏死营养体病原菌灰霉菌后,与野生型相比,突变体株系叶片的病斑水渍化更严重,有更多的细胞死亡和活性氧积累。以上结果表明WRKY75在植物抵御病原菌入侵过程中可能发挥重要作用。(2)克隆了VpPR10.8基因上游-1496bp的启动子序列,对启动子序列进行分析发现含有2个WRKY结合位点(W-box),序列为TTGACC,分别位于基因上游-194bp和-1317bp处。酵母单杂交实验表明VpWRKY75转录因子能特异性结合VpPR10.8启动子中翻译起始位点上游-194bp处的W-box作用元件。同时双荧光素酶实验发现VpWRKY75转录因子能结合VpPR10.8启动子,正调控其活性。对‘无核白’葡萄愈伤组织进行外源激素SA和MeJA处理,发现均能诱导VpWRKY75和VpPR10.8的表达。(3)构建了葡萄WRKY75的过表达和CRISPR/Cas9基因编辑载体,并转入欧洲葡萄‘无核白’胚性细胞中,对其进行抗性筛选。目前已经得到了过表达转基因抗性葡萄植株以及分化的敲除转基因愈伤组织。对部分过表达转基因葡萄苗及敲除转基因抗性愈伤组织进行检测,鉴定出两个阳性过表达转基因葡萄株系。更多还原

【Abstract】 Grape downy mildew is a major oomycete disease that seriously harms grape production.The pathogen(Plasmopara viticola)causing grape downy mildew is a kind of obligate parasitic oomycetes,and there are significant differences in resistance to P.viticola among different grape varieties/species.European grape(Vitis vinifera L.)is susceptible to P.viticola,while the wild grape in China shows resistance to P.viticola.WRKY transcription factors have been confirmed to be involved in plant defense response in recent years,therefore,mining disease resistance-related genes in Chinese wild grape to improve the European grape is greatly significant for grape breeding.In the early stage of the study,our lab screened a transcription factor VpWRKY75 induced by grape downy mildew from Chinese wild grape ’Liuba-8’,and predicted the downstream target gene PR10.8 which possibly interacting with.In this study,VpWRKY75 homologous gene AtWRKY75 Arabidopsis mutant and grape calli were used to analyze the preliminary disease resistance function of WRKY75 and its response to plant hormones,and then the interaction between VpWRKY75 and VpPR10.8 promoter were verified by dual-luciferase assay and yeast one-hybrid experiment.Finally,overexpression and knockout of WRKY75 gene were carried out by Agrobacterium tumefaciens-mediated grape genetic transformation system in order to obtain transgenic grape plants.The main results are as follows:(1)The AtWRKY75 homologous to VpWRKY75 mutated in Arabidopsis was inoculated with different pathogens to analyze the disease resistance function.After inoculating with Phytophthora capsici belonging to the oomycetes as P.viticola,compared with the wild type,the mutant lines had larger chlorotic lesions,more relative biomass and more cell death.After inoculating with Pseudomonas syringae pv.Tomato DC3000,the mutant Arabidopsis leaves showed more sensitive phenotype than the wild type: the chlorosis of leaves was more serious,the number of bacteria was significantly increased,but the programmed cell death and hydrogen peroxide were significantly less.After inoculating Botrytis cinerea,the mutant Arabidopsis showed larger necrotic lesions,more cell death and active oxygen accumulation.The above results suggest that WRKY75 may play an important role in plant defense against pathogen invasion.(2)The promoter sequence of upstream-1496 bp of VpPR10.8 gene was cloned and analyzed.It was found that there were two WRKY binding sites(W-box)in the sequence,and the motif sequence is TTGACC,which located at the upstream-194 bp and-1317 bp of translation initiation site,respectively.Yeast one-hybrid experiment showed that the VpWRKY75 transcription factor could specifically bind to the W-box cis-element at the upstream-194 bp of the translation initiation site in the VpPR10.8 promoter.The dual-luciferase assay also showed that VpWRKY75 transcription factor could bind to the VpPR10.8 promoter and positively regulate its activity.The calli of ’Thompson Seedless’ was treated with exogenous hormones SA and MeJA,it was found that the expression of VpWRKY75 and VpPR10.8 were up-regulated.(3)The overexpression vector and CRISPR/Cas9 gene editing vector of WRKY75 were constructed and transferred into European grape ’Thompson seedless’ embryogenic cells for resistance screening.At present,over-expressed transgenic resistant grape plants and differentiated knockout transgenic calli have been obtained.Some overexpressed transgenic grape seedlings and knockout transgenic resistant calli were detected,two positive overexpression transgenic grape lines were identified.更多还原