摘要
【目的】热激蛋白(heat shock protein,Hsp)是生物体遭受不利环境胁迫时迅速产生的一类抗逆蛋白,可帮助生物机体度过不利环境。本研究旨在探究Hsp 90 AB在中华按蚊Anopheles sinensis生长发育、杀虫剂胁迫和溴氰菊酯抗性中的作用机制,为深入研究该基因的作用机制及中华按蚊的防控提供理论依据。【方法】基于中华按蚊转录组和基因组数据,采用RT-PCR克隆AsHsp 90 AB的全长cDNA序列,并利用生物信息学对该基因序列进行分析;利用RT-qPCR检测AsHsp 90 AB在中华按蚊溴氰菊酯敏感品系(WX-LS)和抗性品系(WX-LR)不同发育阶段(卵、1-4龄幼虫、雌蛹、雄蛹、雌成蚊和雄成蚊)和雌成蚊不同组织(唾液腺、中肠、马氏管、脂肪体、体壁和卵巢)中的表达模式;利用幼虫浸渍(敏感基线法)半致死中浓度(median lethal concentration,LC 50)(0.0016 mg/L)溴氰菊酯和WHO接触桶法(0.05%溴氰菊酯药膜)分别胁迫WX-LS的4龄幼虫和3日龄雌成蚊,利用RT-qPCR检测AsHsp 90 AB的表达达量;通过显微注射法利用RNAi技术沉默中华按蚊WX-LR雌蛹中AsHsp 90 AB,利用RT-qPCR检测AsHsp 90 AB的表达量,观察统计RNAi后48 h时WHO接触桶法0.05%溴氰菊酯药膜处理后雌成蚊的半数击倒时间(median knockdown time,KT 50)、击倒率和存活率。【结果】克隆出中华按蚊AsHsp 90 AB(GenBank登录号:PP405608)2678 bp的全长cDNA序列,开放阅读框长2154 bp,编码718个氨基酸,预测蛋白质理论分子量和等电点分别为81.65 kD和4.94,具有典型的Hsp90家族基因的特征基序,含有MEEVD基序,为细胞质型。系统进化分析上显示,中华按蚊AsHsp90AB与按蚊属Anopheles的Hsp90s聚为一支,再与库蚊属Culex的Hsp90s聚在一起,说明它们之间的亲缘关系较近。AsHsp 90 AB在WX-LS和WX-LR整个发育阶段持续表达,在WX-LS中雄成蚊和雌成蚊卵巢中表达量最高;在WX-LR 4龄幼虫中高表达和雌成蚊触角中表达量最高。溴氰菊酯胁迫后中华按蚊WX-LS 4龄幼虫和3日龄雌成蚊中AsHsp 90 AB表达量分别上调了1.37~2.61倍和1.91~2.58倍,而被击倒的雌成蚊AsHsp 90 AB表达量下调了72.51%。RNAi抑制了中华按蚊WX-LR雌蛹中AsHsp 90 AB的表达,RNAi后24,48和72 h时AsHsp 90 AB的表达量比对照组ds EGFP显著降低,分别下调44.00%,61.09%和55.95%。注射ds AsHsp 90 AB的中华按蚊WX-LR雌成蚊在0.05%溴氰菊酯药膜处理后10 min时开始被击倒,ds EGFP对照组雌成蚊是30 min时开始才被击倒,在0.05%溴氰菊酯药膜处理1 h期间的击倒率逐渐升高,两组的击倒率差异显著。ds EGFP对照组雌成蚊的KT 50为(50.000±1.667)min,ds AsHsp 90 AB处理组雌成蚊的KT 50值为(30.000±0.600)min。在0.05%溴氰菊酯药膜处理后转移至恢复桶恢复24 h时沉默AsHsp 90 AB的WX-LR雌成蚊的存活率比ds EGFP对照组的显著升高了24.5%。【结论】AsHsp 90 AB在WX-LR中高表达,沉默该基因后中华按蚊在溴氰菊酯胁迫下死亡率提高。本研究为AsHsp 90 AB在中华按蚊生长发育和溴氰菊酯抗性中的潜在功能提供有价值的参考。
【Aim】Heat shock proteins(Hsps)are a kind of anti-stress proteins that are rapidly produced when organisms are under adverse environmental conditions,which can help them survive in adverse environments.This study aims to explore the mechanism of Hsp 90 AB in the growth and development of Anopheles sinensis under insecticide stress and in the deltamethrin resistance,laying a foundation for further study of the mechanism of Hsp 90 AB and the prevention and control of An.sinensis.【Methods】Based on the transcriptome and genome data of An.sinensis,the full-length cDNA sequence of AsHsp 90 AB was cloned by using RT-PCR and analyzed by bioinformatics.The expression profiles of AsHsp 90 AB in both deltamethrin-susceptible strain(WX-LS)and deltamethrin-resistant strain(WX-LR)at different developmental stages(egg,1st-4th instar larvae,female pupa,male pupa,female adult and male adult),and in different female adult tissues(salivary gland,midgut,Malpighian tubules,fat body,integument and ovary)of An.sinensis were detected by RT-qPCR.The 4th instar larva and 3-day-old female adult of WX-LS of An.sinensis were stressed with the median lethal concentration(LC 50)(0.0016 mg/L)of deltamethrin using the larval immersion method(baseline susceptibility assay)and the WHO contact bioassay method(0.05% deltamethrin-coated bottles),respectively,and the expression level of AsHsp 90 AB was detected by RT-qPCR.After the AsHsp 90 AB silence by RNAi with microinjection method in WX-LR female pupa,the expression level of AsHsp 90 AB was detected by RT-qPCR.The median knockdown time(KT 50),knockdown rate and survival rate of female adult treated with deltamethrin by 0.05% deltamethrin-coated bottles via the WHO contact bioassay method at 48 after RNAi were observed and calculated.【Results】The full-length cDNA sequence 2678 bp of AsHsp 90 AB(GenBank accession no.:PP405608)in An.sinensis was cloned,with an open reading frame of 2154 bp in length encoding 718 amino acids.The predicted theoretical molecular weight and isoelectric point of AsHsp90AB were 81.65 kD and 4.94,respectively.AsHsp90AB has the characteristic motif of the Hsp90 family and contains the MEEVD motif as a cytoplasmic type.Phylogenetic analysis showed that AsHsp90AB of An.sinensis clustered together with the Hsp90s from Anopheles and then with the Hsp90s from Culex,indicating that they were closely related.AsHsp 90 AB was expressed during the whole developmental stage in both WX-LS and WX-LR,and was highly expressed in adult males and the ovaries of adult females of WX-LS.AsHsp 90 AB exhibited high expression in the 4th instar larva and the highest expression level in female adult antennae of WX-LR.The expression levels of AsHsp 90 AB in the 4th instar larva and 3-day-old female adult of WX-LS of An.sinensis after exposure to deltamethrin stress were upregulated by 1.37-fold-2.61-fold and 1.91-fold-2.58-fold,respectively,while that in the knocked down female adult was downregulated by 72.51%.The expression of AsHsp90AB in female pupa of WX-LR of An.sinensis was inhibited by RNAi and its expression level significantly decreased at 24,48 and 72 h after RNAi by 44.00%,61.09% and 55.95%,respectively,as compared with that in the ds EGFP control group.The female adult of WX-LR of An.sinensis injected with ds AsHsp 90 AB began to be knocked down at 10 min after 0.05% deltamethrin-coated bottle treatment,while that in the ds EGFP control group began to be knocked down at 30 min after 0.05% deltamethrin-coated bottle treatment.The knockout rate gradually increased during 1 h of 0.05% deltamethrin-coated bottle treatment,and the knockout rates of the two groups were significantly different.The KT 50 value of female adult in the ds EGFP control group was(50.000±1.667)min and that in the ds AsHsp 90 AB treatment group was(30.000±0.600)min.The survival rate of female adult of WX-LR of An.sinensis after 0.05% deltamethrin-coated bottle treatment and transferred to the recovery bucket for 24 h after recovery was significantly increased by 24.5% as compared with that in the ds EGFP control group.【Conclusion】AsHsp 90 AB was highly expressed in WX-LR of An.sinensis,and the mortality of An.sinensis under the application of deltamethrin was increased when AsHsp 90 AB was silenced.This study provides a valuable reference for the potential function of AsHsp 90 AB in the growth and development as well as the deltamethrin resistance of An.sinensis.
作者
司风玲
幸晓清
李泉润
陈斌
SI Feng-Ling;XING Xiao-Qing;LI Quan-Run;CHEN Bin(Chongqing Key Laboratory of Vector Control and Utilization,College of Life Sciences,Chongqing Normal University,Chongqing 401331,China)
出处
《昆虫学报》
北大核心
2025年第11期1547-1558,共12页
Acta Entomologica Sinica
基金
重庆市教委科学技术委员会青年项目(KJQN202000535)
重庆市科学技术委员会基础与前沿研究计划(面上)项目(cstc2019jcyj-msxmX0595)
重庆师范大学基金项目(20XLB016)。
关键词
中华按蚊
热激蛋白基因
溴氰菊酯
杀虫剂胁迫
杀虫剂抗性
RNAI
Anopheles sinensis
heat shock protein gene
deltamethrin
insecticide stress
insecticide resistance
RNAi
