适用于玉米螟虫蜕DNA提取方法的筛选与验证

doi:10.6048/j.issn.1001-4330.2025.02.014

新疆农业科学 ›› 2025, Vol. 62 ›› Issue (2): 379-385.DOI: 10.6048/j.issn.1001-4330.2025.02.014

适用于玉米螟虫蜕DNA提取方法的筛选与验证

孙健博¹^,²(), 丁新华², 贾尊尊², 高国文¹^,², 付开赟², 吐尔逊·阿合买提², 安尼瓦尔·库尔班¹(), 郭文超²()

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

收稿日期:2024-08-05 出版日期:2025-02-20 发布日期:2025-04-17
通信作者: 安尼瓦尔·库尔班(1970-),男,新疆乌鲁木齐人,副教授,硕士生导师,研究方向为农业害虫综合防治,(E-mail)1834238969@qq.com;
郭文超(1966-),男,河北人,研究员,博士,硕士生/博士生导师,研究方向为农业害虫综合防治,(E-mail) gwc1966@163.com
作者简介:孙健博(1997-),男,河南周口人,硕士研究生,研究方向为农业昆虫与害虫防治,(E-mail)282276511@qq.com
基金资助:
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)

Screening and verification of DNA extraction methods suitable for corn borer molting

SUN Jianbo¹^,²(), DING Xinhua², JIA Zunzun², GAO Guowen¹^,², FU Kaiyun², Tuerxun Ahemaiti², Anniwaer Kuerban¹(), GUO Wenchao²()

1. College of Agriculture, Xinjiang Agricultural University, Urumqi 830052, China
2. 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

Received:2024-08-05 Published:2025-02-20 Online:2025-04-17
Supported by:
National Natural Science Foundation of China“Population Differentiation and Environmental Driving Mechanism of Interspecific Competition between Asian Corn Borer and European Corn Borer in Xinjiang”(31960538);Maize Industry Technology System of Xinjiang Uygur Autonomous Region(XORS);Agricultural Science and Technology Innovation and Stability Special Project of Xinjiang Academy of Agricultural Sciences“Key Technology Research and Development of Agricultural Diseases“Pests and Weeds and Biosafety Prevention and Control”(xjnkywdzc-2023004)

摘要/Abstract

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。

关键词: 玉米螟; 幼虫蜕; DNA提取; 试剂盒法; 蛋白酶法

Abstract:

【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.

Key words: corn borer; larval exuviate; DNA extraction; kit method; protease method

中图分类号:

S436.41

孙健博, 丁新华, 贾尊尊, 高国文, 付开赟, 吐尔逊·阿合买提, 安尼瓦尔·库尔班, 郭文超. 适用于玉米螟虫蜕DNA提取方法的筛选与验证[J]. 新疆农业科学, 2025, 62(2): 379-385.

SUN Jianbo, DING Xinhua, JIA Zunzun, GAO Guowen, FU Kaiyun, Tuerxun Ahemaiti, Anniwaer Kuerban, GUO Wenchao. Screening and verification of DNA extraction methods suitable for corn borer molting[J]. Xinjiang Agricultural Sciences, 2025, 62(2): 379-385.

图/表 6

参考文献 19

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步骤 Procedure	试剂盒法 DNAiso Reagent Kit method(KM)	蛋白酶法 Protease method(PM)	CTAB法 Cetyltrimethylammonium Bromide method(CM)
(1)研磨裂解 Grinding cracking	加入150 μL的提取液,DNAiso Reagent,滴至样品上,用灭菌后的配套研钵充分研磨	加60 μL提取液滴至样品上,用灭菌后的配套研钵充分研磨	加入1.5 mL提取液滴至样品上,用灭菌后的配套研钵充分研磨。8 000 r/min、4℃离心1 min取上清液
(2)洗脱富集 Elution Enrichment	加入150 μL的提取液,DNAiso Reagent,室温静置5 min。12 000r/min、4℃离心10 min	用60 μL提取液清洗研磨组织,后将匀浆转移至200 μL Ep管中	将上述上清液1经过硅胶模离心吸附柱洗脱后得到虫蜕粗DNA
(3)酶解 Enzymolysis	无	摇匀后放置于温育仪内(65℃,15 min,95℃,10 min)	使用蛋白酶K和RNA酶对虫蜕DNA进行温育过夜酶解,得酶解液
(4)抽提 Extraction	离心后,吸取上清液转移至新Ep管中,加入150 μL无水乙醇,反复颠倒,将混合液加入硅胶模离心吸附柱中。12 000 r/min、4℃离心2 min	无	将酶解液依次经氯仿-异戊醇(24∶1)、等体积的饱和酚和氯仿-异戊醇(24∶1)进行抽提处理
(5)沉淀 Precipitation	离心弃废液,向吸附柱中加入150 μL的75%乙醇。12 000 r/min、4℃离心2 min,重复2次	无	添加0.5倍其体积的异丙醇,混匀,于冰上沉淀20 min,9 000 r/min离心25min,弃上清,得沉淀1
(6)洗涤 Washing	离心后弃废液,空管离心12 000 r/min、4℃离心2 min	无	向沉淀1中加400 μL 75%乙醇,12 000 r/min离心7 min,弃上清。重复该操作1次
(7)溶解 Dissolution	离心弃废液,敞开硅胶模离心吸附柱,室温静置3 min。加入30 μL灭菌ddH₂O在室温静置2 min。12 000 r/min、4℃离心2 min。后于-20℃保存备用。提取过程共计耗时1h	产物储存于-20℃保存备用。提取过程共计耗时40 min	加入30 μL ddH₂O对DNA进行溶解,即得DNA。后于-20℃保存备用。提取过程共计耗时13 h

步骤 Procedure	试剂盒法 DNAiso Reagent Kit method(KM)	蛋白酶法 Protease method(PM)	CTAB法 Cetyltrimethylammonium Bromide method(CM)
(1)研磨裂解 Grinding cracking	加入150 μL的提取液,DNAiso Reagent,滴至样品上,用灭菌后的配套研钵充分研磨	加60 μL提取液滴至样品上,用灭菌后的配套研钵充分研磨	加入1.5 mL提取液滴至样品上,用灭菌后的配套研钵充分研磨。8 000 r/min、4℃离心1 min取上清液
(2)洗脱富集 Elution Enrichment	加入150 μL的提取液,DNAiso Reagent,室温静置5 min。12 000r/min、4℃离心10 min	用60 μL提取液清洗研磨组织,后将匀浆转移至200 μL Ep管中	将上述上清液1经过硅胶模离心吸附柱洗脱后得到虫蜕粗DNA
(3)酶解 Enzymolysis	无	摇匀后放置于温育仪内(65℃,15 min,95℃,10 min)	使用蛋白酶K和RNA酶对虫蜕DNA进行温育过夜酶解,得酶解液
(4)抽提 Extraction	离心后,吸取上清液转移至新Ep管中,加入150 μL无水乙醇,反复颠倒,将混合液加入硅胶模离心吸附柱中。12 000 r/min、4℃离心2 min	无	将酶解液依次经氯仿-异戊醇(24∶1)、等体积的饱和酚和氯仿-异戊醇(24∶1)进行抽提处理
(5)沉淀 Precipitation	离心弃废液,向吸附柱中加入150 μL的75%乙醇。12 000 r/min、4℃离心2 min,重复2次	无	添加0.5倍其体积的异丙醇,混匀,于冰上沉淀20 min,9 000 r/min离心25min,弃上清,得沉淀1
(6)洗涤 Washing	离心后弃废液,空管离心12 000 r/min、4℃离心2 min	无	向沉淀1中加400 μL 75%乙醇,12 000 r/min离心7 min,弃上清。重复该操作1次
(7)溶解 Dissolution	离心弃废液,敞开硅胶模离心吸附柱,室温静置3 min。加入30 μL灭菌ddH₂O在室温静置2 min。12 000 r/min、4℃离心2 min。后于-20℃保存备用。提取过程共计耗时1h	产物储存于-20℃保存备用。提取过程共计耗时40 min	加入30 μL ddH₂O对DNA进行溶解,即得DNA。后于-20℃保存备用。提取过程共计耗时13 h

操作步骤 Operation procedure	提取液 Extract	虫蜕 Moult
操作步骤 Operation procedure	提取液 Extract	C(ng/μL)	A260_nm/A280_nm
试剂盒法 DNAiso Reagent Kit method (KM)	a	0.01±0^b	1.35±0.01^b
	b	0.01±0^b	1.32±0.01^b
	c	8.46±0.22^a	1.86±0.01^a
	d	0.01±0^b	1.34±0.01^b
蛋白酶法 Protease method(PM)	a	0.01±0.02^b	0.91±0.01^a
	b	23.03±0.01^a	0.86±0.02^b
	c	0.01±0.01^b	0.77±0.02^b
	d	0.01±0^a	1.31±0.01^a
CTAB法 Cetyltrimethylammonium Bromide method (CM)	a	0.01±0^a	1.02±0.04^c
	b	0.01±0^a	1.18±0.09^b
	c	0.01±0^a	1.10±0.02^b
	d	0.01±0^a	1.33±0.01^a

操作步骤 Operation procedure	提取液 Extract	虫蜕 Moult
操作步骤 Operation procedure	提取液 Extract	C(ng/μL)	A260_nm/A280_nm
试剂盒法 DNAiso Reagent Kit method (KM)	a	0.01±0^b	1.35±0.01^b
	b	0.01±0^b	1.32±0.01^b
	c	8.46±0.22^a	1.86±0.01^a
	d	0.01±0^b	1.34±0.01^b
蛋白酶法 Protease method(PM)	a	0.01±0.02^b	0.91±0.01^a
	b	23.03±0.01^a	0.86±0.02^b
	c	0.01±0.01^b	0.77±0.02^b
	d	0.01±0^a	1.31±0.01^a
CTAB法 Cetyltrimethylammonium Bromide method (CM)	a	0.01±0^a	1.02±0.04^c
	b	0.01±0^a	1.18±0.09^b
	c	0.01±0^a	1.10±0.02^b
	d	0.01±0^a	1.33±0.01^a

试验对象 Experimental subject	提取液 Pyrolysis liquid	试剂盒法 DNAiso Reagent Kit method	蛋白酶法 Protease method	CTAB法 Cetyltrimethyl ammonium Bromide method
玉米螟虫体 Corn borer body	a	+	-	/
	b	+	+	/
	c	+	-	+
	d	/	/	/
玉米螟虫蜕 Corn borer molting	a	/	/	/
	b	/	+	/
	c	+	/	/
	d	/	/	/

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 methods suitable for corn borer molting

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[1]	郭文超, 贾尊尊, 丁新华, 叶晓琴, 付开赟, 吐尔逊·阿合买提, 王小武, 乔小燕, 孙建博. 新疆荒漠绿洲生态区亚洲玉米螟和欧洲玉米螟的种间竞争取代研究综述[J]. 新疆农业科学, 2024, 61(S1): 1-11.
[2]	孙健博, 吴莉莉, 贾尊尊, 叶晓琴, 丁新华, 付开赟, 吐尔逊·阿合买提, 王哲, 李亚文, 付文君, 艾尔肯娜依·买买提江, 安尼瓦尔·库尔班, 郭文超. 新疆伊犁河谷玉米主要害虫田间一喷多防药效评价[J]. 新疆农业科学, 2024, 61(S1): 12-18.
[3]	蓝陈仪航, 姚禹博, 周俊祥, 付开赟, 丁新华, 尹晓辉, 刘文, 王娜, 郭文超, 邓建宇. 亚洲玉米螟性信息素鉴定及其应用研究进展[J]. 新疆农业科学, 2023, 60(7): 1614-1622.
[4]	袁洁, 丁新华, 贾尊尊, 付开赟, 吐尔逊·阿合买提, 郭文超. 氮素含量对亚洲玉米螟个体发育及种群增殖的影响[J]. 新疆农业科学, 2022, 59(6): 1458-1465.
[5]	丁新华, 王志慧, 贾尊尊, 付开赟, 吐尔逊·阿合买提, 杨杰, 郭文超. 新疆主栽玉米品种不同生育期抗螟性鉴定及评价[J]. 新疆农业科学, 2022, 59(5): 1180-1188.
[6]	袁洁, 丁新华, 付开赟, 贾尊尊, 阿尔孜姑丽·肉孜, 吐尔逊·阿合买提, 郭文超. 氮素含量对亚洲玉米螟取食及选择性影响[J]. 新疆农业科学, 2022, 59(5): 1196-1202.
[7]	丁新华, 王小武, 付开赟, 贾尊尊, 吐尔逊・阿合买提, 郭文超. 温度、相对湿度对亚洲玉米螟Ostrinia furnacalis (Guenée)越冬幼虫化蛹及羽化的影响[J]. 新疆农业科学, 2022, 59(11): 2668-2674.
[8]	库尔班妮萨·奥布力喀斯木, 王俊, 王志慧, 丁新华, 贾尊尊, 付开赟, 阿尔孜姑丽·肉孜, 吐尔逊·阿合买提, 何江, 冯宏祖, 郭文超. 新疆玉米产区47个主栽玉米品种抗虫性评价[J]. 新疆农业科学, 2021, 58(7): 1197-1206.
[9]	支昊宇, 王智慧, 王兴仔, 丁新华, 贾尊尊, 郭文超, 姜卫华. 亚洲玉米螟的抗药性监测及双酰胺类药剂抗性靶标分子检测[J]. 新疆农业科学, 2021, 58(4): 690-699.
[10]	王小武, 丁新华, 付开赟, 刘芳慧, 张惠菊, 吐尔逊, 郭文超. 作物布局对玉米螟种群发生与危害的影响[J]. 新疆农业科学, 2020, 57(7): 1330-1337.
[11]	王志慧, 丁新华, 贾尊尊, 付开赟, 吐尔逊·阿合买提, 何江, 郭文超. 基于增效剂与杀虫剂协同作用的玉米螟超低量喷雾防治技术[J]. 新疆农业科学, 2020, 57(2): 311-318.
[12]	尹鹏,丁新华,付开赟,吐尔逊·阿合买提,何江,郭文超. 玉米螟优势捕食性天敌控害效益评价[J]. 新疆农业科学, 2018, 55(4): 712-718.
[13]	解玉梅;刘芳慧;丁新华;李翠梅;付开赟;何江;吐尔逊·阿合买提;阿卜杜热伊木·阿卜杜热西提;李鹏发;许建军;郭文超. 秸秆储放方式对越冬代玉米螟幼虫化蛹及羽化的影响[J]. , 2017, 54(3): 505-512.
[14]	丁新华;王娜;王小武;付开赟;吐尔逊·阿合买提;何江;杨健;付文君;郭文超. 小型无人机超低量喷雾防治玉米螟田间药效评价[J]. , 2017, 54(3): 479-488.
[15]	阿依克孜, 丁新华, 付开赟, 吐尔逊·阿合买提, 何江, 朱晓华, 班小莉, 古丽夏提, 丁爱琴, 郭文超. 新疆玉米产区三个玉米螟地理种群发育起点、有效积温测定与比较研究[J]. 新疆农业科学, 2017, 54(10): 1863-1874.