Preliminary Stuay On Regulation Of Vtg Gene Expression In Scylla Paramamosain

Vitellogenin(vtg)is a precursor of Vitellin(Vn or Vt),which is the main component of egg yolk proteins.Vtg provides amino acids,vitamins,fats,carbohydrates and other nutrients for embryos and larvae,and plays a crucial role in the maturation of oocyte and production of eggs in aquatic animals.In this study,the 5’-regulatory region sequence of Scylla paramamosain vtg(spvtg)gene was cloned.Meanwhile,on the basis of the of Spvtg 3’non-coding region,miRNAs that may regulate Spvtg were searched.Furthermore,the expression regulation mechanisms of Spvtg at the transcriptional level and post-transcriptional level were analyzed by using transient transfection and dual luciferase reporter gene assays.The major results were stated below:1)864 bp 5’flanking region of Spvtg was obtained by Genome Walking and Tail PCR methods.2)Bioinformatics analysis predicted the position of the transcription initiation site(TSS)that was defined as 1 and the putative core promoter region in the region of-40～+16bp..The promoter of Spvtg has a TATA box between-24～-31 bp upstream of the transcription initiation site.3)To identify the promoter activity and the core promoter region of Spvtg,two reporter plasmids(one containing the complete 864 bp 5’-upstream region was named pGL-vtg-rl;the other one removing core promoter region was named pGL-vtg-rr1)were constructed and transfected into HEK293T cells.The activity of pGL-vtg-rl was significantly higher than pGL-vtg-rrl and negative control(pGL3-Basic,plasmid without inserts)(p<0.05).4)Five luciferase plasmids with different length deletion fragments were successfully constructed.Five constructed plasmids all had high activities.Among them,the activity of pGL-vtg-r4 was lower than that of pGL-vtg-r5(p<0.05).Mutation analysis of transcription factor binding sites between the two shortest deletion fragments found that REV-ErbA may be an important negative transcription factor which regulates the expression of Spvtg.5)Bioinformatics analysis suggested that there is putative binging site for miR-34 in the 3’UTR of Spvtg.After the vtg3’ UTR-WT/vtg3’UTR-MUT double luciferase vector was constructed,it was co-transfected with miR-34-mimics/miR-34-mimicsNC into HEK293T cells.The results showed that the activity of vtg 3’UTR-WT was significantly decreased by miR-34-mimics(p<0.05),the inhibition of Spvtg by miR-34 was verified.6)After injection with the miR-34 agomir/miR-34 antagomir into S.paramamosain,the relative expression of miR-34 and vtg in ovarian and hepatopancreas was detected.The results showed that after injection with miR-34 agomir,the relative expression of miR-34 increased significantly(p<0.05),and the relative expression of vtg decreased significantly(p<0.05);after injection with miR-34 antagomir,the relative expression of miR-34 decreased significantly(p<0.05),and the relative expression of vtg increased significantly(p<0.05).These results confirmed the role of miR-34 in regulating Spvtg expression.