基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术

›› 2015, Vol. 58 ›› Issue (5): 579-586.doi:

基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术

陈苗苗¹, 郭荣³, 张金良⁴, 万方浩^1,2,5, 杨锦¹, 张桂芬^1,2,*

(1. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193; 2. 农业部外来入侵生物预防与控制研究中心, 北京 100193; 3. 全国农业技术推广服务中心, 北京 100026; 4. 北京市植物保护站, 北京 100029; 5. 青岛农业大学农学与植物保护学院, 山东青岛 266109）

出版日期:2015-05-20 发布日期:2015-05-20
作者简介:陈苗苗, 女, 1988年10月生, 山东济宁人, 硕士研究生, 研究方向为入侵生物学, E-mail: chenmiaomiao_2012@163.com

Rapid identification of Aleyrodes proletella (Hemiptera: Aleyrodidae), a new invasive whitefly species in mainland China, based on SS-COI marker

CHEN Miao-Miao¹, GUO Rong³, ZHANG Jin-Liang⁴, WAN Fang-Hao^1,2,5, YANG Jin¹, ZHANG Gui--Fen^1,2,*

(1. State Key Laboratory for the Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Center for Management of Invasive Alien Species, Ministry of Agriculture, Beijing 100193, China; 3. National AgroTechnical Extension and Service Centre, Beijing 100026, China; 4. Beijing Plant Protection Station, Beijing 100029, China; 5. College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao, Shandong 266109, China)

Online:2015-05-20 Published:2015-05-20

摘要/Abstract

摘要： 【目的】针对粉虱类害虫种类多、体型微小、形态相似、难以准确快速识别的问题，以新入侵我国大陆的甘蓝粉虱 Aleyrodes proletella (L.)为靶标，以田间常见的其他10种/隐种粉虱为参照，采用基于线粒体DNA细胞色素C氧化酶亚基I (mitochondrial DNA cytochrome c oxidase subunit I, mtDNA COI) 基因的种特异性 (species-specific COI, SS-COI) PCR方法，研究其快速分子检测技术。【方法】利用mtDNA COI基因通用型引物LCO-1490/HCO-2198获得甘蓝粉虱及其他常见粉虱的COI序列，根据测序结果设计种特异性SS-COI引物1对（APZYJF/APZYJR），其扩增片段大小为384 bp，同时对该对引物的种特异性及灵敏性进行检测。【结果】种特异性检验结果显示，该对引物仅对甘蓝粉虱的mtDNA COI基因具有扩增效果，对我国常见的其他种类的粉虱包括温室粉虱 Trialeurodes vaporariorum (Westwood)、柑橘粉虱 Dialeurodes citri (Ashmead)、螺旋粉虱 Aleurodicus disperses (Russell)、双钩巢粉虱 Paraleyrodes pseudonaranjae Martin、非洲伯粉虱 Bemisia afer (Priesner et Hosny)以及烟粉虱B. tabaci (Gennadius)5个隐种（MED, Asia I, Asia II 1, Asia II 3和China 1）等不具有交叉反应扩增能力。灵敏性检验结果显示，该对引物不仅对不同性别的成虫具有良好的扩增效能，对2-4龄若虫甚至单粒卵或单头初孵若虫亦具有同样的扩增能力，其最低检测阈值为14.00±0.37 pg/μL（相当于1/25 600头雌性成虫）。【结论】该技术体系完全可用于甘蓝粉虱的快速准确识别及检测监测，对有效阻截其进一步扩张蔓延意义重大。

关键词: 甘蓝粉虱, 隐种, 种特异性SSCOI引物, 快速鉴定, 分子检测

Abstract: 【Aim】 Morphological identification of Aleyrodes proletella(L.) is limited by small size, high degree of similarity to related species and polymorphism. This study aims to develop a technique based on mitochondrial DNA cytochrome c oxidase subunit I (mtDNA COI) gene sequence for the rapid identification of A. proletella, an invasive species newly found in mainland China. 【Methods】 The fragments of mtDNA COI gene of A. proletella and ten other whitefly species or cryptic species were amplified and sequenced using COI gene universal primers LCO-1490/HCO-2198. One pair of species-specific COI (SS-COI) primers, APZYJF and APZYJR, was designed. The target fragment amplified by the SS-COI primers (APZYJF/APZYJR) is 384 bp in length. The species specificity and sensitivity of the primer pair were tested. 【Results】 The species specificity of the primer pair was validated using ten other whitefly species and cryptic species, i.e., Trialeurodes vaporariorum (Westwood), Dialeurodes citri (Ashmead), Aleurodicus disperses (Russell), Paraleyrodes pseudonaranjae Martin, Bemisia afer (Priesner et Hosny), and five cryptic species of B. tabaci (Gennadius) (MED, Asia I, Asia II 1, Asia II 3 and China 1). All A. proletella specimens were successfully detected, and no cross reaction with other whitefly species or cryptic species was observed. The method was tested using individual male or female adult, individual 2nd instar, 3rd instar or red-eyed nymph, and even one grain of egg or single newly emerged 1st instar nymph, and proved to be applicable for all of these life stages. Sensitivity test results demonstrated that successful amplification could be obtained with the concentration of template DNA as low as 14.00±0.37 pg/μL, equal to 1/25 600 of a whole female adult of A. proletella. 【Conclusion】 The SS-COI method developed here provides a quick, simple and reliable molecular technique for the identification and monitoring of A. proletella, which would be useful in intercepting and blocking the further spreading of this new invasive whitefly species.

Key words: Aleyrodes proletella, cryptic species, SS-COI primers, rapid identification, molecular detection

陈苗苗, 郭荣, 张金良, 万方浩, 杨锦, 张桂芬. 基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术[J]. , 2015, 58(5): 579-586.

CHEN Miao-Miao, GUO Rong, ZHANG Jin-Liang, WAN Fang-Hao, YANG Jin1, ZHANG Gui--Fen. Rapid identification of Aleyrodes proletella (Hemiptera: Aleyrodidae), a new invasive whitefly species in mainland China, based on SS-COI marker[J]. , 2015, 58(5): 579-586.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://www.insect.org.cn/CN/

http://www.insect.org.cn/CN/Y2015/V58/I5/579

[1]	杨春红, 郭建洋, 褚栋, 程登发, 万方浩. 漆酶-1基因在烟粉虱MEAM1隐种和温室粉虱中的表达模式比较[J]. , 2018, 61(9): 1019-1028.
[2]	郭磊, 黄新意, 梁延坡, 刘想想, 褚栋. 海南烟粉虱田间种群隐种鉴定及对溴氰虫酰胺和氟吡呋喃酮的敏感性检测[J]. , 2018, 61(2): 209-217.
[3]	姚志文, 杨帆, 吴月坤, 魏洪义, 陆宴辉. 放置时间和条件对空僵蚜中蚜虫和寄生蜂DNA检出率的影响[J]. , 2017, 60(6): 691-698.
[4]	应丰泽, 孙艳春, 潘李隆, 洪越, 刘树生. 两个入侵烟粉虱隐种MEAM1与MED对番茄黄曲叶病毒水平传播能力的比较[J]. , 2015, 58(8): 872-880.
[5]	黄振, 陈韶萍, 谢婧, 郭琼霞. 应用种特异性PCR技术快速鉴定辣椒实蝇[J]. , 2015, 58(4): 460-466.
[6]	刘国霞, 高长生, 付海滨, 褚栋. 烟粉虱Q与B隐种mtCOI基因的遗传变异及其对利用CAPS标记进行隐种鉴别的影响[J]. , 2014, 57(10): 1238-1244.
[7]	白润娥，王雄雅，李静静，刘晓华，熊大斌，李冬兵 . 烟粉虱MED隐种铁蛋白基因克隆、不同发育阶段和吡虫啉胁迫下的表达及原核表达[J]. , 2013, 56(7): 738-746.
[8]	田虎, 李小凤, 万方浩, 张桂芬, 张金良. 利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧[J]. , 2013, 56(6): 689-696.
[9]	龙楚云, 郭建洋, 万方浩. 烟粉虱MED隐种保幼激素酯酶cDNA片段克隆、序列分析及在不同发育阶段的表达[J]. , 2013, 56(3): 234-242.
[10]	杨春红, 彭露, 郭建洋, 严盈, 万方浩, 王进军. 烟粉虱MEAM1隐种漆酶-1基因全长cDNA克隆、序列分析与组织表达[J]. , 2013, 56(2): 111-119.
[11]	秦丽, 王佳, 邴孝利, 刘树生. 利用mtCOI PCR-RFLP技术鉴定中国境内九个烟粉虱隐种[J]. , 2013, 56(2): 186-194.
[12]	郭雅洁, 梁沛, 高希武. 烯啶虫胺、毒死蜱和高效氯氰菊酯对烟粉虱地中海隐种hsp70 mRNA的诱导表达[J]. , 2013, 56(1): 29-38.
[13]	姚晶, 郭晓军, 王甦, 王昱超, 罗晨, 张帆, 李绍勤. 利用TaqMan等位基因技术鉴定烟粉虱MEAM1和MED隐种[J]. , 2013, 56(1): 98-103.
[14]	刘循, 万方浩, 张桂芬. 可用于黑刺粉虱快速鉴定的SCAR分子标记技术[J]. , 2009, 52(8): 895-900.