适用于玉米螟虫蜕DNA提取方法的筛选与验证  

Screening and verification of DNA extraction methodssuitable for corn borer molting

作　　者：孙健博 丁新华[2] 贾尊尊 高国文 付开赟 吐尔逊·阿合买提[2] 安尼瓦尔·库尔班[1] 郭文超[2] SUN Jianbo;DING Xinhua;JIA Zunzun;GAO Guowen;FU Kaiyun;Tuerxun Ahemaiti;Anniwaer Kuerban;GUO Wenchao(College of Agriculture,Xinjiang Agricultural University,Urumqi 830052,China;Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis,Ministry of Agriculture and Rural Affairs/Xinjiang Key Laboratory of Agricultural Biosafety/Institute of Plant Protection,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,China)

机构地区：[1]新疆农业大学农学院,乌鲁木齐830052 [2]新疆农业科学院植物保护研究所/农业农村部西北荒漠绿洲作物有害生物综合治理重点试验室/新疆农业生物安全重点试验室,乌鲁木齐830091

出　　处：《新疆农业科学》2025年第2期379-385,共7页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金项目“新疆同域亚洲玉米螟和欧洲玉米螟种间竞争取代的种群分化与环境驱动机制”(31960538);新疆维吾尔自治区玉米产业技术体系(XORS);新疆农业科学院农业科技创新稳定专项“农业病虫草害与生物安全防控关键技术研发”(xjnkywdzc-2023004)。

摘　　要：【目的】新疆伊犁河谷是亚洲玉米螟和欧洲玉米螟混合发生区,采用一种对玉米螟无损伤的提取方法,高效建立纯系种群,准确分离鉴定亚洲玉米螟和欧洲玉米螟,为后期开展生物学生态学研究提供科学依据。【方法】以玉米螟虫蜕为材料,建立一套无形态特征损伤、经济适用、操作简单、快速且能满足后续PCR扩增检测技术的DNA提取方法。基于筛选试剂盒法(KM)、蛋白酶法(PM)、CTAB法(CM)等常用DNA提取方法,提取玉米螟虫蜕DNA、靶标COI序列扩增、琼脂糖凝胶检测。【结果】虫蜕有效的提取方法为处理3:蛋白酶法,其中蛋白酶法提取虫蜕的DNA浓度最高,为(23.03±0.01)ng/μL,但其纯度不高,不利于后续PCR扩增。处理3提取虫蜕的DNA浓度平均值为(8.46±0.22)ng/μL,符合提取要求,纯度较高为1.86,满足后续PCR扩增试验。【结论】处理3与蛋白酶法可以用于大批量提取玉米螟虫蜕gDNA,种群鉴定具有对玉米螟无损伤、成本低、步骤简单、避免氯仿和异丙醇伤害等优点,处理3较蛋白酶法有较高的扩增效率。【Objective】Ostrinia furnacalis is the main pest that causes maize yield reduction in Xinjiang.Ostrinia nubilalis(Hubern)and Ostrinia furnacalis(Guenee)are sister species that are difficult to be identified by morphology.The Yili River Valley in Xinjiang is a mixed occurrence area of the Eurasian corn borer,so it is very important to carry out accurate isolation and identification of the Eurasian corn borer in this area.a non-destructive extraction method for the corn borer will be used to efficiently establish a pure line population,which is very important for the later biological ecology research.【Methods】The purpose of this paper is to establish a DNA extraction method without morphological damage,economical,simple,rapid and can meet the subsequent PCR amplification detection technology.Based on the common DNA extraction methods such as screening kit method(KM),protease method(PM) and CTAB method(CM),the DNA extraction,target COI sequence amplification and agarose gel detection of corn borer molt were carried out.【Results】The screening results showed that the effective extraction method for the molt was treatment 3:protease method.The DNA concentration of the molt extracted by the protease method was the highest,which was(23.03±0.01)ng/μL,but its purity was not high,which was not conducive to subsequent PCR amplification.The average concentration of DNA extracted by treatment 3 was(8.46±0.22)ng/μL,meeting the extraction requirements,and the purity was 1.86,meeting the subsequent PCR amplification experiment.【Conclusion】The treatment 3 and protease method mentioned in this paper can be used to extract gDNA from corn borer molt in large quantities,and at the same time to carry out population identification and subsequent experimental research.Both of them have the advantages of no damage to corn borer,low cost,simple steps,avoiding the harm of chloroform and isopropanol to human body,and it has higher amplification efficiency than the protease method.

关 键 词：玉米螟 幼虫蜕 DNA提取 试剂盒法 蛋白酶法 

分 类 号：S436.41[农业科学—农业昆虫与害虫防治]