梨树腐烂病菌致病力缺陷突变体的筛选和侧翼序列分析  

Screening of pathogenicity defective mutants and analysis of flanking sequence in Valsa pyri

作　　者：袁洪波 侯珲[1] 周增强[1] 涂洪涛[1] 王丽[1] YUAN Hongbo;HOU Hui;ZHOU Zengqiang;TU Hongtao;WANG Li(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences,Zhengzhou 450009,Henan,China)

机构地区：[1]中国农业科学院郑州果树研究所,郑州450009

出　　处：《果树学报》2021年第11期1966-1974,共9页Journal of Fruit Science

基　　金：新疆兵团重大科技项目(2019AA004);中国农业科学院科技创新工程(CAAS-ASTIP);中央级科研院所基本科研业务费专项(1610192020105)。

摘　　要：【目的】从梨树腐烂病菌T-DNA插入突变体库中筛选鉴定致病缺陷突变体,并分离致病相关基因。【方法】利用农杆菌介导方法转化梨树腐烂病菌,获得504个T-DNA插入转化子,对其中250个转化子的致病力进行筛选。利用TAIL-PCR对致病力显著降低的突变体的T-DNA插入位点的侧翼序列进行扩增和分析。【结果】与野生型菌株相比,4个转化子(T8、T12、T43和T75)在梨果实和枝条上致病力显著降低。扩增获得T8、T43和T75的T-DNA插入位点的特异侧翼序列,测序后经Blast比对序列结果显示,转化子T8、T43和T75 T-DNA分别插在含MSF结构域的蛋白基因、Zds1基因和假定的谷氨酸合成酶基因。【结论】构建了农杆菌介导的梨树腐烂病菌T-DNA插入突变体库,从中筛选获得4个致病力缺陷的突变体,并分析了致病力缺陷突变体的T-DNA插入位点的侧翼序列,为下一步梨树腐烂病菌致病基因的克隆和功能研究奠定了基础。【Objective】Pear is one of the most important fruits worldwide and China is a major production area. However, certain microbial pathogens and insects pose a great threat to pear production.Among pear tree diseases, pear Valsa canker disease caused by Valsa pyri is one of the most serious threats to pear growth in East Asia including China. V. pyri can invade host tissue wounded by injury in the bark. Following V. pyri infection, pear trees exhibit reddish-brown, water-soaked, softened barks,and even plant death in some cases, resulting in severe economic losses. In the important pear planting regions of China, like Northwest China, pear Valsa canker disease has become more and more common and destructive in recent years, which greatly affects the development of pear industry. However, it is difficult to control the disease by chemical application, because V. pyri can penetrate systemically in xylem and phloem tissues and most active compounds of fungicides are not able to protect the internal phases of the tree trunk. In addition, this pathogen can infect the host plant at any time of the year.Therefore, the functional characterization of virulence genes and pathogenic mechanism of V. pyri are urgently needed, which would help us to make comprehensive prevention and controlling strategy. However, most studies on V. pyri conducted to date have focused on the identification of this pathogen, investigation of disease regularity and fungicide selection, rather than on the actual basis for its pathogenesis.The aim of this study is to identify pathogenicity defective mutants from a T-DNA insertion mutant library of V. pyri and to analyze pathogenicity-related genes, which are expected to provide a better understanding of the pathogenic mechanism of V. pyri.【Methods】We used an approach to the Agrobacterium tumefaciens-mediated transformation(ATMT) of V. pyri with a plasmid vector encoding the green fluorescent protein(GFP) and hygromycin resistance(Hyg) genes. Transformants were screened by placing on PDA plates

关 键 词：梨树腐烂病菌 突变体 致病力 侧翼序列 

分 类 号：S661.2[农业科学—果树学]