苹果树腐烂病菌GTP-环化水解酶II基因敲除载体构建及其突变体的表型分析  

Construction of Knockout Vector of GTP Cyclohydrolase II Gene and Mutant's Biological Characteristics of Valsa mali

作　　者：宋娜[1] 戴青青 宋娜[1] 黄丽丽[1] 韩青梅 

机构地区：[1]西北农林科技大学植物保护学院、旱区作物逆境生物学国家重点实验室,陕西杨凌712100

出　　处：《中国农业科学》2014年第15期2980-2989,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31171796);公益性行业(农业)科研专项(201203034);高等学校博士学科点基金(20120204110002);高等学校学科创新引智计划(B07049)

摘　　要：【目的】利用反向遗传学方法构建苹果树腐烂病菌(Valsa mali)中表达GTP-环化水解酶II基因Vmgtp1的敲除载体,通过同源重组的方法进行基因敲除分析目标基因的功能,为全面解析苹果树腐烂病菌致病的分子机制奠定基础,并为有效控制苹果树腐烂病的方法技术和药剂研制提供理论依据。【方法】通过对笔者实验室苹果树腐烂病菌转录组数据的分析比对得到GTP-环化水解酶II基因Vmgtp1(暂命名),该基因在接种5 d后,在病组织中表现出一定的上调表达,推测该基因可能与苹果树腐烂病菌致病相关。采用Double-joint PCR方法,将目标基因Vmgtp1的上游片段UP、下游片段DOWN及筛选标记潮霉素磷酸转移酶基因(hph)进行片段融合。对得到的PCR产物及质粒pHIG2RHPH2-GFP-GUS进行双酶切,通过T4连接酶,将片段UP-HPH-DOWN连接到质粒pHIG2RHPH2-GFP-GUS上,转化到JM109菌株中,挑取阳性克隆,提取质粒,得到含有hph的Vmgtp1敲除载体,再利用PEG介导的遗传转化获得抗潮霉素的突变体,然后用4对引物对突变体进行PCR检测及Southern Blot验证得到敲除突变体,最后对敲除突变体及野生型菌株03-8进行菌落颜色、菌落大小、繁殖体产生情况等表型分析和接种在离体苹果烫伤枝条上观察病斑大小的致病性检测,对检测数据用SPSS软件进行差异显著性分析。【结果】通过上述方法成功构建了表达GTP-环化水解酶II基因的Vmgtp1敲除载体,并利用PEG介导的遗传转化方法在苹果树腐烂病菌中进行了转化,共获得101个能够在含潮霉素PDA培养基上生长的突变菌株,经过PCR及Southern Blot对101个突变菌株进行分析验证,得到1个敲除突变体,编号为ΔVmgtp1-90。在PDA培养基上,敲除突变体ΔVmgtp1-90与野生型菌株03-8相比,ΔVmgtp1-90菌落平均生长速率为11.33 mm·d-1,03-8平均生长速率为24.67 mm·d-1,突变体菌落生长速率明显变慢,颜色变浅,气生菌丝稀疏;ΔVmgtp1-90�[Objective] The objective of this study is to construct the knockout vector of gene Vmgtp1 encoding GTP cyclohydrolase II using the strategy of reverse genetics, and analyze the gene preliminary function by homologous recombination, so as to lay a foundation for the comprehensive analysis of pathogenic molecular mechanisms in Valsa mali and provide a theoretical basis for the technology and pharmaceutical research of apple rot disease. [Method] Based on the analysis of the transcriptome data of V. mali, the gene Vmgtp1 （tentatively named） was obtained. After inoculating for 5 days, the expression in the infected tissue was increased. So it is predicted that Vmgtp1 may be related with pathogenicity in V. mali. Using Double-joint PCR method, the up and downstream fragment of Vmgtp1 and the selectable marker hygromycin-phosphotransferase gene （hph） to one fragment were connected. The PCR product and plasmid pHIG2RHPH2-GFP-GUS were double-digested, connected with T4 ligase and transformed into JM109. Then the positive plasmid was transformed into V. mali through PEG-mediated genetic transformation. Next, the hgromycin-resistant mutant strains which can grow normally on PDA medium containing hygromycin were selected. The selected homologous recombinant mutants were then confirmed by PCR with four pairs of primers and Southern Blot. At last, the phenotypes of wild-type strain 03-8 and mutant were analyzed through observation colony color, colony size and propagulum production, the pathogenicities were tested by observing the lesion sizes on the apple limbs which inoculated with wild-type strain 03-8 and mutant. The significance of differences was analyzed by the software of SPSS.[Result]The knockout vector of gene Vmgtp1 encoded GTP cyclohydrolase II using the above-mentioned method was constructed successfully and 101 mutant strains with PEG-mediated transformation were obtained. Only one homologous recombination mutant ΔVmgtp1-90 was obtained after PCR and Southern Blot testing the 101 mutant strains. Compar

关 键 词：苹果黑腐皮壳 同源重组 PEG 致病性 

分 类 号：S436.611[农业科学—农业昆虫与害虫防治]