Functional Analysis Of The Promoters Of Mih Gene And Chh Gene In Scylla Paramamosain

Crustacean hyperglycemic hormone(CHH)family plays an important role in the regulation of metamorphosis development,reproduction,molting,blood glucose balance and internal osmotic pressure balance in crustacean.Molt-inhibiting hormone(MIH)and CHH are two important members of CHH family.In this study,the 5’ flanking region sequences of mih gene and chh gene were cloned by Genome walking and Tail-PCR.Then bioinformatics analysis was maked to obtained sequence.At last,recombinant plasmids construction,transient transfection and dual-luciferase reporter assays were used to analyze the function of mih gene and chh gene.The results are as follows:1)The 5’ flanking region sequence of mih gene cloned by Genome walking and Tail-PCR is 2024 bp starting from the translation start site(ATG).Forecast analysis results by the bioinformatics software showed that the transcription start site is located at 207 bp upstream of start codon ATG,and TATA box is located at 33 bp upstream of transcription start site.Potential transcription factor binding sites include Ap-1,Sp1,NF-1,Oct-1,Sox-2,RAP1,C/EBPalp,NF-κappaB,GATA-1,and so on.There are two CpG islands,located at-25 bp — +183 bp and-1451 bp —-1316 bp respectively.2)The 5’ flanking region sequence of chh gene cloned by Genome walking and Tail-PCR is 657 bp starting from the translation start site(ATG).Forecast analysis results by the bioinformatics software showed that the transcription start site is located at 74 bp upstream of start codon ATG,and TATA box is located at 31 bp upstream of transcription start site.Potential transcription factor binding sites include Oct-1,C/EBPalp,GATA-1,Sp1,Ap-1,NF-κappaB,NF-1,RAP1,HNF-3,and so on.There is no CpG island in the 5’ flanking region sequence of chh gene.3)Activity analysis of deletion fragments of mih gene promoter in HEK293 T and Sf9 cells showed that there were binding sites of potential positive transcription factors between-1350 bp—-889 bp and between-308 bp—-26 bp.The point mutation and activity analysis experiment about binding site of transcription factor NF-κB between-1350 bp—-889 bp suggested that NF-κB may play a driving role in the regulation of mih gene expression.The point deletion and activity analysis experiment about binding site of transcription factor RAP1 between-308 bp—-26 bp suggested that RAP1 may be an important positive transcription factor to regulate mih gene expression.4)Activity analysis of luciferase reporter constructs including polymorphism CA repeat of mih gene promoter showed that the activity of the fragment including CA(n=11)was the highest,and the activity of the fragment including CA(n=10)was higher than that of the fragment including CA(n=12).5)Activity analysis of deletion fragments of chh gene promoter in HEK293 T and Sf9 cells showed that there were binding sites of potential negative transcription factors between-286 bp—-46 bp and-583 bp—-388 bp,and that there were binding sites of potential positive transcription factors between-388 bp—-286 bp.