云南省三带喙库蚊对DDT和溴氰菊酯抗性群体的击倒抗性基因突变分析

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (6): 536-540.

云南省三带喙库蚊对DDT和溴氰菊酯抗性群体的击倒抗性基因突变分析

姜进勇^1,², 陈辉莹², 周红宁¹, 马雅军^2,^*()

1云南省寄生虫病防治所,云南省虫媒病毒研究中心,云南省虫媒传染病防控研究重点实验室,云南公共卫生与疾病防控协同创新中心,普洱 665099
2 第二军医大学热带医学与公共卫生学系热带传染病学教研室,上海 200433

收稿日期:2017-04-26 出版日期:2017-12-30 发布日期:2018-01-10
通讯作者: 马雅军
基金资助:
国家自然科学基金（No.81371848,No.30660160,No.81160357）

Analysis of knowdown resistance gene mutation in Culex tritaeniorhynchus resistant to DDT and deltamethrin in Yunnan Province, China

Jin-yong JIANG^1,², Hui-ying CHEN², Hong-ning ZHOU¹, Ya-jun MA^2,^*()

1 Yunnan Institute of Parasitic Diseases, Yunnan Provincial Center of Arborvirus Research, Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research, Yunnan Provincial Collaborative Innovation Center for Public Health and Disease Prevention and Control, Puer 665099, China
2 Department of Tropical Infectious Disease, Second Military Medical University, Shanghai 200433, China

Received:2017-04-26 Online:2017-12-30 Published:2018-01-10
Contact: Ya-jun MA
Supported by:
Supported by the National Natural Science Foundation of China （No.81371848, No.30660160, No.81160357）

摘要/Abstract

摘要：

目的分析经接触DDT和溴氰菊酯敏感性测定的三带喙库蚊（Culex tritaeniorhynchus）的抗性（存活）与敏感（死亡）表型样本的击倒抗性基因（knockdown resistance, kdr）序列,阐明其抗性表型与kdr基因突变的关系。方法收集云南昭阳、芒市、江城和孟连等地的三带喙库蚊经接触DDT和溴氰菊酯测定后死亡和存活的个体,分别提取单蚊基因组DNA,PCR扩增kdr部分基因片段,测定和分析序列,统计抗性与敏感表型个体中kdr突变的基因型和频率,采用χ²检验分析kdr基因突变与抗性表型的相互关系。结果共获得411条三带喙库蚊kdr基因序列,检测结果显示,在1014位点存在突变,等位基因有2种,即野生型TTA/L和突变型TTT/F;基因型共3 种,分别为野生型纯合子L/L,突变型纯合子F/F,以及野生型/突变型杂合子L/F,频率分别为96.35%（396/411）、0.73%（3/411）和2.92%（12/411）。在接触DDT的抗性与敏感表型个体中,突变基因型频率分别为3.42%（4/117）和3.88%（4/103）;在接触溴氰菊酯的抗性与敏感个体中,突变基因型频率为3.96%（4/101）和3.33%（3/90）。接触DDT和溴氰菊酯的三带喙库蚊抗性与敏感表型与kdr基因型频率无相关性（χ² = 0.034,P > 0.05;χ² = 0.053,P > 0.05）。结论云南省三带喙库蚊中发现kdr新的等位基因L1014F,但频率较低。该蚊对DDT和溴氰菊酯产生抗性与kdr基因突变未见相关性。

关键词: 三带喙库蚊, 击倒抗性, 基因突变

Abstract:

Objective To detect the knockdown resistance（kdr） gene mutations in Culex tritaeniorhynchus resistant or susceptible to DDT and deltamethrin, in order to elucidate the relationship between the resistant phenotype and kdr mutations.Methods C.tritaeniorhynchus mosquitoes were captured in Zhaoyang, Mang Shi, Jiang Cheng and Meng Lian in Yunnan Province, and exposed to DDT and deltamethrin, respectively.The resulting alive and dead ones were counted.Genomic DNA was extracted by individual mosquitoes.The kdr gene was amplified by PCR and analyzed.The frequency of kdr genotypes was estimated in resistant and susceptible samples.The relationship between kdr genotypes and resistant phenotypes was analyzed by Chi-square test.Results A total of 411 mosquitoes were genotyped for the kdr gene.Mutation was detected at codon 1014, yielding two alleles, the wild-type TTA/L and the mutant TTT/F, and three genotypes, the wild-type homozygote L/L （frequency, 96.35%; 396/411）, the mutant homozygote F/F （0.73%; 3/411） and the wild/mutant heterozygous L/F （2.92%; 12/411）.The frequencies of mutant genotype in DDT-resistant and -susceptible individuals were 3.42% （4/117） and 3.88% (4/103), respectively; while those in deltamethrin-resistant and -susceptible individuals were 3.96% (4/101) and 3.33% （3/90）, respectively.Chi-square test revealed that there was no correlation between kdr genotype frequency and the resistance phenotypes to either DDT （χ² = 0.034, P > 0.05） or deltamethrin （χ² = 0.053, P > 0.05）.Conclusion We found a novel L1014F mutant allele for kdr, albeit with a low frequency.There is no correlation between kdr gene mutation and the resistance phenotypes to either DDT or deltamethrin.

Key words: Culex tritaeniorhynchus, Knockdown resistance, Gene mutation

中图分类号:

R384.112

姜进勇, 陈辉莹, 周红宁, 马雅军. 云南省三带喙库蚊对DDT和溴氰菊酯抗性群体的击倒抗性基因突变分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 536-540.

Jin-yong JIANG, Hui-ying CHEN, Hong-ning ZHOU, Ya-jun MA. Analysis of knowdown resistance gene mutation in Culex tritaeniorhynchus resistant to DDT and deltamethrin in Yunnan Province, China[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 536-540.

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引物名 Primers	序列 (5′→3′) Sequences (5′→3′)	片段长度/bp Product length/bp
CT3	TGCTGTGCGGAGAGTGGATC	800
CT6	TTTCATTCAACTTGGCTCTG
TRI5	CTTCACCGACTTCATGCACTC	200
TRI6	GATTTTGGACAAAAG CAAGGC

杀虫剂 Insecticide	地点 Sampling site	表型 Phenotype	蚊虫数 No.	野生型纯合子(L/L) Wild type (L/L)			突变型杂合子(L/F) Heterozygote (L/F)			突变型纯合子(F/F) Mutant genotype (F/F)
杀虫剂 Insecticide	地点 Sampling site	表型 Phenotype	蚊虫数 No.	蚊虫数 No.mosquitoes	频率/% Frequency/%		蚊虫数 No.mosquitoes		频率/% Frequency/%	蚊虫数 No.mosquitoes		频率/% Frequency/%
DDT	昭阳Zhaoyang	R	45	41		91.11	4	8.89		0	0
	昭阳Zhaoyang	S	55	51		92.73	3	5.45		1	1.82
	芒市Mangshi	R	26	26		100	0	0		0	0
	芒市Mangshi	S	35	35		100	0	0		0	0
	江城Jiangcheng	R	13	13		100	0	0		0	0
	江城Jiangcheng	S	6	6		100	0	0		0	0
	孟连Menglian	R	33	33		100	0	0		0	0
	孟连Menglian	S	7	7		100	0	0		0	0
	小计Subtotal	R	117	113		96.58	4	3.42		0	0
	小计Subtotal	S	103	99		96.12	3	2.91		1	0.97
溴氰菊酯 Deltamethrin	昭阳Zhaoyang	R	23	21		91.30	1	4.35		1	4.35
	昭阳Zhaoyang	S	24	21		87.5	2	8.33		1	8.32
	芒市Mangshi	R	36	35		97.22	1	2.78		0	0
	芒市Mangshi	S	44	44		100	0	0		0	0
	江城Jiangcheng	R	17	17		100	0	0		0	0
	江城Jiangcheng	S	9	9		100	0	0		0	0
	孟连Menglian	R	25	24		96.00	1	4.00		0	0
	孟连Menglian	S	13	13		100	0	0		0	0
	小计Subtotal	R	101	97		96.04	3	2.97		1	0.99
	小计Subtotal	S	90	87		96.67	2	2.22		1	1.11