高温胁迫下绿豆象内参基因筛选及其Hsp70基因的表达特性  

Identification of reference genes and expression analysis of heat shock protein 70 gene in the Chinese bean weevil Callosobruchus chinensis under high temperature stress

作　　者：郑海霞[1] 张天浩 张敏[1] 张仙红[1] 贾彬良 Zheng Haixia;Zhang Tianhao;Zhang Min;Zhang Xianhong;Jia Binliang(College of Plant Protection,Shanxi Agricultural University,Taigu 030801,Shanxi Province,China;Shanxi Farmland Quality Monitoring and Protection Center,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西农业大学植物保护学院,太谷030801 [2]山西省耕地质量监测保护中心,太原030001

出　　处：《植物保护学报》2022年第3期799-808,共10页Journal of Plant Protection

基　　金：山西省自然基金面上项目(20210302124166);国家重点研发计划(2021YFD1600603-01);财政部和农业农村部国家现代农业产业技术体系(CARS-08-G10)。

摘　　要：为明确Hsp70蛋白在绿豆象Callosobruchus chinensis高温耐受性中的作用,该研究筛选并克隆β-actin、α-tubulin、β-tubulin、EF1-α、GAPDH、Hsc70、AK和RPL40八个候选内参基因,采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术测定其在绿豆象雌雄成虫体内的表达量,通过geNorm、BestKeeperr和NormFinde软件对这8个候选内参基因的表达稳定性进行评价,选择最适宜的内参基因对不同高温胁迫下绿豆象体内Hsp70基因的表达特性进行测定。结果表明,8个候选基因引物均有良好的特异性和引物扩增效率,引物扩增效率介于91.6%~108.1%之间。高温胁迫下,β-tubulin基因较其他7个候选基因有较小的平均变异度、稳定值和标准差,为最适宜的内参基因。39、42和45℃高温处理1 h后绿豆象雌雄成虫体内Hsp70表达水平较对照显著上调,当处理时间延长至3 h后,Hsp70基因的相对表达量较对照也有不同程度上调,但差异不显著,表明绿豆象Hsp70基因可以响应高温胁迫,Hsp70蛋白在绿豆象热耐受过程中发挥着重要作用。In order to clarify the role of heat shock protein 70(Hsp70)in high temperature tolerance of Chinese bean weevil Callosobruchus chinensis,eight candidate reference genes were identified and cloned from C.chinensis,namelyβ-actin,α-tubulin,β-tubulin,EF1-α,GAPDH,Hsc70,AK and RPL40.The expression levels of these genes were detected in both male and female adults of C.chinensis by quantitative real-time PCR(qPCR).The expression stabilities of these candidate genes were evaluated by the geNorm,BestKeeper and NormFinder programs and the most suitable reference gene was selected.The expression levels of Hsp70 gene in C.chinensis adult under different heat stresses regimes were measured by using the suitable reference gene.The results indicated that the primers of the eight candidate genes showed high amplification efficiency and specificity(91.6%-108.1%).Under temperature stress,β-tubulin had the lowest mean variability value,stability value and standard deviation.It was the most stably expressed reference gene in C.chinensis.It was significantly upregulated after exposure to 39,42 and 45℃for one hour in female and male of C.chinensis.Under temperature stress for 3 h,the expression of Hsp70 was upregulated slightly but there was no significant difference compared to the control.These results indicated that the Hsp70 gene could respond to heat stress,and Hsp70 might play an important role in heat tolerance of C.chinensis.

关 键 词：绿豆象 温度 胁迫 热激蛋白70 表达谱 

分 类 号：S433.5[农业科学—农业昆虫与害虫防治]