应用锁式探针结合斑点杂交技术检测甜瓜细菌性叶斑病  

Development of Padlock Probe Combined with Dot-Blot Hybridization Based Methods for Detection of Bacterial Spot of Melon Leaves

作　　者：徐瑞 胡白石[2] 田艳丽[2] 黄艳宁 谢进 曹亮 彭斯文 朱校奇 XU Rui HU BaiShi TIAN YanLi HUANG YanNing XIE Jin CAO Liang PENG SiWen ZHU XiaoQi(Institute of Agricultural and Biological Resources Utilization, Hunan Academy of Agricultural Sciences, Changsha 410125 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095)

机构地区：[1]湖南省农业生物资源利用研究所,长沙410125 [2]南京农业大学植物保护学院,南京210095

出　　处：《中国农业科学》2017年第4期679-688,共10页Scientia Agricultura Sinica

基　　金：国家"863"计划(2012AA101501);国家公益性行业(农业)科研专项(201303117)

摘　　要：【目的】甜瓜细菌性叶斑病是危害甜瓜的重要种传细菌病害,是由丁香假单胞杆菌流泪病致病变种甜瓜菌株(Pseudomonas syringae pv.lachrymans)引起的。论文旨在建立高效、快捷、操作简单的检测技术以防止此病原菌传播。【方法】以Gen Bank公布的甜瓜细菌性叶斑病菌的甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因序列为靶标,设计甜瓜细菌性叶斑病菌特异性锁式探针,建立锁式探针与斑点杂交技术结合的检测体系。以保存的目的菌株为材料,提取DNA作为模板进行锁式探针连接反应、酶切反应和扩增反应,并将锁式探针连接反应、酶切反应和扩增反应的反应温度和反应时间分别进行优化。利用优化的锁式探针连接反应、酶切反应和扩增反应分别对健康的甜瓜种子、带有甜瓜细菌性叶斑病的甜瓜种子、无菌水及25株其他参试菌株进行检测,测定该体系的特异性。将甜瓜细菌性叶斑病菌的DNA按10倍梯度稀释,依次稀释为1 ng·μL^(-1)、100 pg·μL^(-1)、10 pg·μL^(-1)、1 pg·μL^(-1)、100 fg·μL^(-1)、10 fg·μL^(-1)和1 fg·μL^(-1)作为模板,利用优化的锁式探针连接反应、酶切反应和扩增反应,测定灵敏度。将探针与斑点杂交技术结合建立高通量检测体系,将上述反应过程中的扩增产物固定于尼龙膜上,将锁式探针上Zipcode序列的反向互补序列合成c Zipcode(检测探针),检测探针(c Zipcode)用地高辛标记后与产物进行杂交。锁式探针结合斑点杂交技术分别进行特异性检测和灵敏度测定。探针与斑点杂交技术结合建立的高通量检测体系进行人工模拟种子带菌检测,进一步验证该体系的可靠性。利用建立的高通量检测方法对205份市售疑似带病的甜瓜种子进行检测。【结果】锁式探针特异性测定结果表明,26株甜瓜细菌性叶斑病菌均能得到一条105 bp的特异性条带,而剩余25株参试菌【Objective】 Bacterial spot of melon leaves caused by Pseudomonas syringae pv. lachrymans is distributed widely in the world and inflicts different degrees of damage. P. syringae pv. lachrymans is a typical seed-borne pathogen. The objective of this study is to build effective, commercially viable and convenient detecting technologies and to prevent the spread of this pathogen.【Method】The house-keeping gene of DNA glyceraldehyde-3-phosphate dehydrogenase（GAPDH） was selected as the target gene, the specific Padlock probe was designed for bacterial leaf spot of melon, and a bacterial detection system based on Padlock probe combined with dot-blot hybridization was developed. The target strain was selected as experimental materials, and DNA was extracted as a template for ligation reaction, enzyme-cleavage reaction and amplification reaction. The reaction temperature and reaction time of the ligation reaction, enzyme-cleavage reaction and amplification reaction were optimized. The optimized reactions were tested for healthy melon seeds, melon seeds with bacterial spot of melon leaves, sterile water and 25 experimental strains. In order to determine the sensitivity of Padlock probe, DNA of the target strain was diluted to 1 ng·μL^（-1）, 100 pg·μL^（-1）, 10 pg·μL^（-1）, 1 pg·μL^（-1）, 100 fg·μL^（-1）, 10 fg·μL^（-1） and 1 fg·μL^（-1） as the templates. Sensitivity was determined by optimized ligation reaction, enzyme-cleavage reaction and amplification reaction. Padlock probe combined with dot-blot hybridization was developed, the amplification product in the course of the reaction was fixed on the nylon membrane, the reverse complementary sequence of the Zipcode sequence was synthesized into c Zipcode（detection probe）, the detection probe（c Zipcode） was labeled with digoxigenin and hybridized with the amplified product. Padlock probe combined with dot-blot hybridization techniques were used for specific detection and sensitivity. Artificial infestation seeds were de

关 键 词：甜瓜细菌性叶斑病 锁式探针 斑点杂交 种子 

分 类 号：S436.5[农业科学—农业昆虫与害虫防治]