Molecular mechanism of 20E regulating BmFoxL2-2 in Bombyx mori and expression analysis of SIFoxL2-2 in the testis of Spodoptera litura(Lepidoptera:Noctuidae)
[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 2 500 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 1 μmol/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 6 th 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 2 500 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 of the control group.BmBrc-z1 and BmBrc-z4 were expressed in the testis of larva,prepupa,pupa and adult of B.mori.The expression levels of BmBrc-z1 and BmBrc-z4 in the testis of the 5 th instar larva and 3-day-old pupa were significantly higher than those in the ovary.The expression level of SlFoxL2-2 of S.litura was high in the testis among different tissues,and was significantly higher in the testis and sperm than in the testis membrane of S.litura larvae,suggesting that SlFoxL2-2 also functions in the development of testis,especially in the spermatogenesis.[Conclusion]The expression of BmFoxL2-2 is regulated by BmBRC-Z1 and BmBRC-Z4 through 20E in B.mori,and the function of SlFoxL2-2 in the development of testis may be conserved in lepidopteran insects.
万方数据
维普
