小分子热激蛋白基因HaHSP19.8在棉铃虫抗逆反应中的作用

Role of the small heat shock protein gene HaHSP19.8 in response to stress in Helicoverpa armigera(Lepidoptera:Noctuidae)

【目的】小分子热激蛋白(small heat shock protein,sHSP)在昆虫抵御外界环境压力中至关重要。本研究旨在探究小分子热激蛋白sHSP19.8基因在棉铃虫Helicoverpa armigera生长发育、抵御高温胁迫和对Cry1Ac杀虫蛋白抗性机制中的作用,为更深入探析该基因作用机理及棉铃虫的防治奠定基础。【方法】通过PCR结合RACE克隆棉铃虫sHSP19.8基因序列,利用生物信息学软件对该基因序列进行分析;通过qRT-PCR测定Cry1Ac敏感棉铃虫5龄幼虫在40℃高温下处理1 h和2 h及饲喂含30μg/mL Cry1Ac的人工饲料1 h和2 h后该基因的表达量,并测定抗感Cry1Ac棉铃虫不同发育阶段(1-5龄幼虫、蛹及成虫)和5龄幼虫不同组织(前肠、中肠、后肠、马氏管及表皮)中该基因的表达模式。【结果】获得了棉铃虫sHSP19.8基因的全长cDNA序列,命名为HaHSP19.8(GenBank登录号:XP_021195228.1),长608 bp,开放阅读框长528 bp,编码175个氨基酸残基,具有小分子热激蛋白的典型α-晶体结构域(α-crystallin domain,ACD)。该基因受40℃高温和30μg/mL Cry1Ac杀虫蛋白诱导时在Cry1Ac敏感棉铃虫5龄幼虫中均过量表达;在Cry1Ac敏感棉铃虫整个发育阶段和5龄幼虫各组织中均表达,其中在成虫和5龄幼虫以及5龄幼虫表皮、马氏管和中肠内表达量较高;但是该基因在Cry1Ac抗性品系各个发育阶段和5龄幼虫各组织中表达量相比敏感品系都显著较低。【结论】结果说明HaHSP19.8参与棉铃虫生长发育和生理生化的过程,帮助昆虫抵御外界环境压力,并可能参与到棉铃虫对Cry1Ac的抗性机制中。

【Aim】Small heat shock proteins(sHSPs)play important roles in resisting external environmental pressure in insects.This study aims to explore the functions of a small heat shock protein gene,sHSP19.8,in the growth and development of the cotton bollworm,Helicoverpa armigera,and its role in response to high temperature stress and insecticidal protein,so as to provide a foundation for further exploring its action mechanisms and controlling the cotton bollworm.【Methods】The full-length cDNA sequence of sHSP19.8 gene was cloned from H.armigera by RCR and RACE,and analyzed by bioinformatics software.The expression levels of sHSP19.8 gene in the 5th instar larvae of the Cry1Ac-susceptible cotton bollworm exposed to 40℃for 1 h and 2 h and fed with the artificial diet containing 30μg/mL Cry1Ac for 1 h and 2 h,respectively,were detected by qRT-PCR.Meanwhile,the expression patterns of sHSP19.8 gene in different developmental stages(1st-5th instar larva,pupa and adult)and tissues(foregut,midgut,hindgut,Malpighian tubules and cuticle)of the 5th instar larvae of the Cry1Ac-susceptible and resistant cotton bollworm were also assayed by qRT-PCR.【Results】The full-length cDNA sequence of sHSP19.8 from H.armigera was obtained and named HaHSP19.8(GenBank accession no.:XP_021195228.1).It is 608 bp in length with an open reading frame(ORF)of 528 bp,encoding 175 amino acids.The encoded protein has the typicalα-crystallin domain(ACD).After induction by high temperature of 40℃and 30μg/mL Cry1Ac,HaHSP19.8 showed over-expression in the 5th instar larvae of the Cry1Ac-susceptible cotton bollworm.HaHSP19.8 was ubiquitously expressed in all the examined developmental stages and tissues of the 5th instar larvae of the Cry1Ac-susceptible cotton bollworm,with relatively higher expression levels in adults and the 5th instar larvae,and the cuticle,Malpighian tubules and midgut of the 5th instar larvae.However,HaHSP19.8 showed lower expression levels in all the examined developmental stages and tissues of the 5th instar larvae of the Cry1Ac-resistant cotton bollworm than in those of the Cry1Ac-susceptible cotton bollworm.【Conclusion】The results suggest that HaHSP19.8 may participate in the growth and development of the cotton bollworm to resist the environment stress,and may be involved in the resistance to Cry1Ac.