20E调控家蚕BmFoxL2-2的分子机制及斜纹夜蛾SlFoxL2-2在精巢中的表达分析  

Molecular mechanism of 20E regulating BmFoxL2-2 in Bombyx mori and expression analysis of SlFoxL2-2 in the testis of Spodoptera litura(Lepidoptera:Noctuidae)

作　　者：肖妍虹 裴梦圆 贺智颖 刘邓捷 文靓 余小强 胡启豪 XIAO Yan-Hong;PEI Meng-Yuan;HE Zhi-Ying;LIU Deng-Jie;WEN Liang;YU Xiao-Qiang;HU Qi-Hao(Guangdong Key Laboratory of Insect Developmental Biology and Applied Technology,School of Life Sciences,South China Normal University,Guangzhou 510631,China)

机构地区：[1]华南师范大学生命科学学院,广东省昆虫发育调控与应用研究重点实验室,广州510631

出　　处：《昆虫学报》2024年第5期603-610,共8页Acta Entomologica Sinica

基　　金：华南师范大学青年教师科研培育基金(671676);中国博士后科学基金面上项目(2022M721219)。

摘　　要：【目的】本研究旨在阐明蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone,20E)调节家蚕Bombyx mori转录因子基因BmFoxL2-2表达的分子机制,并分析FoxL2-2在斜纹夜蛾Spodoptera litura精巢中的表达模式。【方法】PCR扩增家蚕5龄幼虫精巢基因组中BmFoxL2-2起始密码子上游约2500 bp的启动子序列,通过生物信息学分析预测其潜在的顺式作用元件(cis-regulatory elements,CREs);分别将pEGFP-BmBRC-Z1,pEGFP-BmBRC-Z2,pEGFP-BmBRC-Z4转录因子表达质粒和pGL3-BmFoxL2-2质粒共转染家蚕BmN细胞,通过双荧光素酶活性实验检测BmBRC蛋白对BmFoxL2-2启动子活性的影响;利用1μmol/L 20E处理BmN细胞4 h时,通过qRT-PCR检测BmFoxL2-2的表达量,检测BmBrc-z1和BmBrc-z4的表达量作为对照;通过qRT-PCR检测BmBrc-z1和BmBrc-z4在家蚕不同发育阶段(幼虫、预蛹、蛹和成虫)精巢中及5龄幼虫和3日龄蛹精巢与卵巢中的表达量;通过生物信息学在斜纹夜蛾基因组数据库鉴定了SlFoxL2-2,利用qRT-PCR检测SlFoxL2-2在斜纹夜蛾6龄幼虫和成虫的精巢与卵巢、6龄幼虫不同组织(脂肪体、中肠、血淋巴、头、表皮和精巢)、不同发育阶段精巢以及6龄幼虫精巢、精子和精巢膜中的表达量。【结果】获得家蚕BmFoxL2-2(Gene ID:101735653)起始密码子上游2500 bp启动子序列,并在序列中预测到13个潜在的BRC蛋白CRE。BmBRC-Z1和BmBRC-Z4可显著上调BmFoxL2-2启动子活性;20E处理BmN细胞4 h时,BmN细胞中BmFoxL2-2,BmBrc-z1和BmBrc-z4的表达量比对照组的显著上调;BmBrc-z1和BmBrc-z4在家蚕幼虫、预蛹、蛹和成虫精巢中均有表达,在5龄幼虫及3日龄蛹精巢中的表达量显著高于卵巢中的。斜纹夜蛾SlFoxL2-2在精巢中有高水平表达,且在精巢和精子中的表达量显著高于在精巢膜中的,可能SlFoxL2-2与精巢发育和精子发生相关。【结论】20E可通过家蚕转录因子BmBRC-Z1和BmBRC-Z4上调BmFoxL2-2的表达,FoxL2-2在调节精巢发育和精子发生中的�【Aim】To clarify the molecular mechanism of 20-hydroxyecdysone(20E)in regulating the expression of the transcription factor gene BmFoxL2-2 of Bombyx mori,and analyze the expression pattern of SlFoxL2-2 in the testis of Spodoptera litura.【Methods】The 2500 bp promoter sequence upstream of the initiation codon of BmFoxL2-2 was cloned from the testis genomic DNA of the 5th instar larva of B.mori by PCR and the potential cis-regulatory elements(CREs)were predicted by bioinformatics tools.pEGFP-BmBRC-Z1,pEGFP-BmBRC-Z2 and pEGFP-BmBRC-Z4 transcription factor expression plasmids and pGL3-BmFoxL2-2 plasmid were co-transfected into the BmN cells and the dual luciferase activity assay was performed to analyze the effects of BmBRCs on the promoter activity of BmFoxL2-2.The expression level of BmFoxL2-2 in the BmN cells at 4 h after treatment with 1μmol/L 20E was determined by qRT-PCR,and the expression levels of BmBrc-z1 and BmBrc-z4 were detected as the control.The expression levels of BmBrc-z1 and BmBrc-z4 in the testis at different developmental stages(larva,prepupa,pupa and adult),and in the testis and ovary of the 5th instar larva and 3-day-old pupa of B.mori were determined by qRT-PCR.SlFoxL2-2 in the genome database of S.litura was identified by bioinformatics and the expression levels of SlFoxL2-2 in the testis and ovary of the 6th instar larva and adult,different tissues(fat body,midgut,haemolymph,head,cuticle and testis)of the 6th instar larva,the testis at different developmental stages,and in the testis,sperm and testis membrane of the 6th instar larva were determined by qRT-PCR.【Results】The about 2500 bp promoter sequence upstream of the initiation codon of BmFoxL2-2(Gene ID:101735653)of B.mori was obtained,and 13 potential CREs of BRC were predicted.BmBRC-Z1 and BmBRC-Z4 significantly up-regulated the promoter activity of BmFoxL2-2,and the expression levels of BmFoxL2-2,BmBrc-z1 and BmBrc-z4 in the BmN cells at 4 h after treatment with 20E were also significantly up-regulated as compared with those

关 键 词：家蚕 精巢发育 蜕皮激素 FoxL2-2 转录调控 

分 类 号：Q969.35[生物学—昆虫学]