多主棒孢SdhB-H278Y突变位点real-time PCR检测体系的建立与应用  

Establishment and Application of Real-time PCR for Detecting SdhB-H278Y Mutation in The SdhB Gene Associated with Corynespora cassiicola in Cucumber

作　　者：朱广雪 阎昱韬 孙炳学 周荣佳 岳圆圆 谢学文[2] 柴阿丽[2] 李磊 李宝聚[2] 石延霞[2] ZHU Guangxue;YAN Yutao;SUN Bingxue;ZHOU Rongjia;YUE Yuanyuan;XIE Xuewen;CHAI Ali;LI Lei;LI Baoju;SHI Yanxia(College of Horticulture,Qingdao Agricultural University,Qingdao 266109,Shandong,China;Institute of Vegetables and Flowers,Chinese Academy of Agriculture Sciences,Beijing 100081,China)

机构地区：[1]青岛农业大学园艺学院,山东青岛266109 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《中国蔬菜》2023年第1期60-67,共8页China Vegetables

基　　金：国家大宗蔬菜产业技术体系项目(CARS-23);国家自然科学基金项目(31972482);中国农业科学院创新工程项目(CAASASTIP-IVFCAAS)。

摘　　要：根据GenBank已登录序列中黄瓜多主棒孢琥珀酸脱氢酶B亚基(SdhB)基因序列差异,针对SdhB-H278Y突变设计特异性引物,建立SdhB-H278Y突变实时荧光定量PCR(real-time PCR)检测体系。结果表明:供试多主棒孢携带SdhBH278Y、SdhB-I280V突变;SdhB-H278Y突变株对啶酰菌胺抗性较强,EC50值为21.47μg·mL^(-1)或>30μg·mL^(-1);建立的real-time PCR检测体系具有良好的线性关系,相关系数R2=0.9929,可特异性检测SdhB-H278Y突变,灵敏度为3.6×10^(-4) ng·μL^(-1),为AS-PCR的10倍。利用携带SdhB-H278Y突变不同比例的基因组DNA对检测体系进行验证,预期值与检测值具有很高的相关性,R^(2)=0.9997;利用该检测体系对山东地区黄瓜棒孢叶斑病病斑中多主棒孢SdhB-H278Y突变株所占比例进行检测,检测结果为0.12%~2.69%。综上,本试验建立的real-time PCR检测体系高效、灵敏、定量,可用于多主棒孢SdhB-H278Y突变的检测,为黄瓜棒孢叶斑病抗性治理提供技术支持。Based on the differences of SdhB gene of Corynespora cassiicola registered in GenBank,specific primers were designed for SdhB-H278Y mutation,and the real-time PCR detection system was established for detecting SdhB-H278Y mutation.The results showed that C.cassiicola carried SdhB-H278Y and SdhB-I280V mutations.The SdhB-H278Y mutants have high resistance to boscalid,the EC50 value of SdhB-H278Y mutants was 21.47μg·mL^(-1)or>30μg·μL^(-1).The established standard curve of detection system existed a favorable linear correlation(R^(2)=0.9929).The detection system can detect the SdhB-H278Y mutation specifically with sensitivity of 3.6×10^(-4) ng·μL^(-1),which was 10 times of AS-PCR.The expected values were highly correlated with the detected values by verifying genomic DNA carrying different mutation ratios of SdhB-H278Y(R^(2)=0.9997).The system could also be used to detect the proportion of SdhB-H278Y mutant in the disease spot of corynespora leaf spot of cucumber,and the detection values were 0.12%-2.69%in Shandong.An efficient,sensitive and quantitative real-time PCR system was established for detecting SdhBH278Y mutation,providing technical support for corynespora leaf spot of cucumber controlling.

关 键 词：黄瓜 多主棒孢 real-time PCR 抗药性 啶酰菌胺 

分 类 号：S436.421.1[农业科学—农业昆虫与害虫防治]