韭菜迟眼蕈蚊小热休克蛋白基因BosHsp23.3的克隆及高温胁迫下的表达与功能分析OA

[Aim]To clarify the role of small heat shock protein(sHsp)sHsp23 in the response of Bradysia odoriphaga to high temperature stress.[Methods]The open reading frame sequence of BosHsp23.3,which was up-regulated in the transcriptome of B.odoriphaga after high temperature stress,was amplified by PCR and subjected to bioinformatic analysis.The expression levels of BosHsp 23.3 in different developmental stages(egg,1st-4th instar larvae,1-5-day-old pupae and adult),different adult tissues(head,thorax and abdomen),and the 4th instar larvae of B.odoriphaga exposed to high temperatures(30,32,34 and 36 ℃)for 1,2,4,6,8,10 and 12 h,followed by recovery at 25 ℃ for 1 and 2 h were quantified using qPCR.RNAi was used to silence BosHsp23.3 in the 4th instar larvae of B.odoriphaga,the expression level of BosHsp23.3 in the 4th instar larvae was determined by qPCR,and their survival rate following short-term heat shock at 38 ℃ was detected.[Results]The open reading frame of BosHsp23.3 in B.odoriphaga was 606 bp in length,encoding 201 amino acids.The encoded protein contained the classical α-crystallin structural domain of small heat shock proteins,suggesting that it belongs to the small heat shock protein family.BosHsp23.3 was the most closely related to sHsps from other dipteran flies and clustered within the same clade.BosHsp23.3 was expressed in all developmental stages of B.odoriphaga,with the lowest expression level in larvae.The expression level of BosHsp23.3 was higher in female adults than in male adults,and was extremely significantly different in the abdomens between female and male adults.High temperature stress induced BosHsp23.3 expression,with the expression levels peaking at 2 h after exposure to 32 and 34 ℃,followed by a decrease with prolonged exposure time.The expression level of BosHsp23.3 under 36 ℃ remained high and stable.After the high temperature stress was removed,the expression level of BosHsp23.3 decreased as the recovery time prolonged.Compared to the control group(fed with the artificial diet containing dsGFP),RNAi-mediated silencing of BosHsp23.3 significantly reduced the survival rate of the 4th instar larvae of B.odoriphaga under 38 ℃ stress.[Conclusion]BosHsp23.3 of B.odoriphaga can respond to high temperature stress and plays an important role in the molecular mechanism of resistance to high temperature stress of B.odoriphaga.