基于种特异性线粒体细胞色素氧化酶Ⅰ引物快速鉴定茄二十八星瓢虫和马铃薯瓢虫  

Rapid identification of Henosepilachna vigintioctopunctata and Henosepilachna vigintioctomaculata based on species-specific mitochondrial cytochrome oxidase Ⅰ primers

作　　者：郭木娟 林妙金 潘广 庾家琪 吕晶 郭威 张亚锋 杨春晓 邱宝利[1] ZHOU Xuguo 潘慧鹏 GUO Mujuan;LIN Miaojin;PAN Guang;YU Jiaqi;LÜ Jing;GUO Wei;ZHANG Yafeng;YANG Chunxiao;QIU Baoli;ZHOU Xuguo;PAN Huipeng(College of Plant Protection,South China Agricultural University/Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province,Guangzhou 510642,China;National Modern Agricultural Industry Science and Technology Innovation Center of Guangzhou,Guangzhou 510520,China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,South China Agricultural University,Guangzhou 510642,China;Department of Entomology,University of Kentucky,Lexington 40546,USA)

机构地区：[1]华南农业大学植物保护学院/广东省生物农药创制与应用重点实验室,广东广州510642 [2]广州国家现代农业产业科技创新中心,广东广州510520 [3]华南农业大学亚热带农业生物资源保护与利用国家重点实验室,广东广州510642 [4]肯塔基大学昆虫学系,肯塔基州列克星敦市40546

出　　处：《华南农业大学学报》2022年第1期59-66,共8页Journal of South China Agricultural University

基　　金：国家重点研发计划(2017YFD0200900);国家自然科学基金(31972269)。

摘　　要：【目的】茄二十八星瓢虫Henosepilachna vigintioctopunctata和马铃薯瓢虫Henosepilachna vigintioctomaculata都是茄科作物上的重要害虫,对茄科作物造成极大的经济损失。这2种瓢虫由于外形非常相似,无法通过体表特征区分,因此很有必要开发一种准确且快速的区分方法。【方法】基于这2种瓢虫线粒体细胞色素氧化酶I(Mitochondrial cytochrome oxidase I,mtCOI)的物种特异性,利用种特异性(Species-specific,SS)PCR引物建立了一种分子鉴定技术,即以茄二十八星瓢虫和马铃薯瓢虫之间的mtCOI基因序列变异为基础,设计了2对SS-mtCOI引物Hvp和Hvm。【结果】用这2对引物进行PCR扩增,均出现物种特异性扩增现象。在不同的DNA质量浓度下对该SS-mtCOI引物进行敏感性检测,结果表明,Hvp引物在茄二十八星瓢虫DNA质量浓度为3.13 mg/L时仍可检测到扩增条带,Hvm引物在马铃薯瓢虫DNA浓度为2.43 mg/L时仍能检测到扩增条带。此外,Hvp和Hvm引物也能准确鉴定马铃薯瓢虫和茄二十八星瓢虫的卵和1龄幼虫,从6个不同省份采集的田间种群也能通过Hvp引物准确鉴定。【结论】这2对SS-mtCOI引物能快速、准确、灵敏地鉴别茄二十八星瓢虫和马铃薯瓢虫。【Objective】Henosepilachna vigintioctopunctata(Fabricius)and Henosepilachna vigintioctomaculata(Motschulsky),two kinds of phytophagous ladybeetles in China,are destructive pests causing great damage to solanaceous plants.They are difficult to distinguish based on external morphological characteristics,and it is therefore necessary to develop a rapid and accurate method to differentiate them.【Method】We established a molecular identification technique using species-specific(SS)PCR primers based on the species-specificity of mitochondrial cytochrome oxidase I(mtCOI)of two ladybeetles.Two pairs of SS-mtCOI primers,Hvp and Hvm,were designed based on sequence variations in the mtCOI gene between H.vigintioctopunctata and H.vigintioctomaculata.【Result】PCR amplifications were conducted using these two primers and both had species-specific amplifications.Sensitivity assays were conducted under different DNA concentrations,and the results showed that Hvp primers for H.vigintioctopunctata had a detectable amplification band at a DNA concentration of 3.13 mg/L,while Hvm primers for H.vigintioctomaculata had a detectable amplification band at a DNA concentration of 2.43 mg/L.The egg or 1st instar of H.vigintioctopunctata and H.vigintioctomaculata could also be accurately differentiated by Hvp and Hvm primers.Furthermore,six field populations of H.vigintioctopunctata collected from six provinces could be authenticated by the Hvp primers.【Conclusion】These two pairs of SS-mtCOI primers can differentiate H.vigintioctopunctata and H.vigintioctomaculata rapidly,accurately and sensitively.

关 键 词：茄二十八星瓢虫 马铃薯瓢虫 SS-mtCOI引物 分子鉴定 敏感性检测 

分 类 号：S433.5[农业科学—农业昆虫与害虫防治]