Preliminary Studies On Screening The Transcription Factors Of VIH Gene And Their Expression In Scylla Paramamosain

Vitellogenesis-inhibiting hormone(VIH)is one of crustacean hyperglycemia hormone(CHH)which plays an important role in the regulation of gonadal development and maturation of crustaceans.Based on the preliminary study on the transcriptional regulation analysis of SpVIH,the promoter activity was re-tested in Sf9 cell,and the key transcription factors were searched by EMSA and BIAcore analysis.In addition,the expression of these transcription factors was analyzed.These results shed light on the mechanism of how these transcription factors regulate VIH and other related genes.The main findings were as follows:1)The promoter activity of SpVIH with seven different deletion fragments(SpVIH-1-7)showed that there were both positive and negative transcription factors binding sites between SpVIH-4 and SpVIH-6.The site-directed mutagenesis of the Oct1 / Oct4 / Sox9 binding site predicted in SpVIH-4 region showed that the transcriptional activity of the SpVIH-4-mut was significantly decreased,which were consistent with the results obtained in the HEK293 FT cells.The results showed that the transcription factor binding site of Oct1 / Oct4 / Sox9 was the key region for regulating the expression of VIH,and might be the important positive transcription factors.2)It was confirmed that the nuclear extract of eyestalk had proteins could bind to the key region of VIH promoter by EMSA experiment,and these specific bindings were dependent on the presence of Oct1 / Oct4 / Sox9 binding sites.3)The results of the EMSA were further validated by BIAcore analysis.It was found that the Oct4 protein was specifically bound to human Anti-Oct4 antibody in the nuclear extract of eyestalk,and the SoxE protein was specifically bound to the human Anti-Sox9 antibody.4)Utilizing the LC-MS/MS system,we identified that the binding proteins recoverd from EMSA and BIAcore contained Oct4 and SoxE subfamily members.From SDS-PAGE results surmising that Oct1 / Oct4 / SoxE might interacted with each other or with other small molecule binding proteins to participate in the regulation of VIH expression.5)Using the biologically active recombinant human Oct4 protein,we found that it could bind to the key promoter region of VIH and showed a concentration-dependent effect,suggesting SpOct4 protein was involved in the regulation of VIH expression.6)The cDNA of the transcription factor SpOct4 gene is 1098 bp encoding 399 amino acids residues,which contains conserved domain POUs and the homologous domain POUh.The results of qRT-PCR showed that the expression of SpOct4 was the highest at the cleavage stage and decreased gradually with embryogenesis.SpOct4 was mainly expressed in the gonads,eyestalk,intestine and heart in mature female crabs,and the expression was significantly different from that of male(P<0.05).During the different stages of ovarian development,SpOct4 was expressed at the highest level in primary vitellogenesis stage(O3)and was significantly higher than other stages(P<0.05).During the testis development,SpOct4 was only expressed in a small amount and no significant difference.Rabbit anti-Oct4 antibody was used in the Western blot detection,and the result showed that it can specifically binds to the corresponding protein of eyestalk and ovary in crabs,which demonstrated the prensent of Oct4 protein.7)According to the SpOct1 EST sequence selected from the transcriptome database,SpOct1 expression was highest in cleavage,5 pairs of appendages and 7 pairs of appendages during embryogenesis(P<0.05).SpOct1 was mainly expressed in the gonads,eyestalk,muscle,intestine and heart in mature female crabs,and the expression was significantly different from that of male(P<0.05).During the different stages of ovarian development,SpOct1 was expressed at the highest level in previtellogenesis stage(O2),primary vitellogenesis stage(O3)and secondary vitellogenesis stage(O4)and was significantly higher than other stages(P<0.05).During the testis development,SpOct1 was only expressed in a small amount and no significant difference.Rabbit anti-Oct1 antibody can specifically bind to a small amount of protein in the eyestalk and ovary,which demonstrated the prensent of Oct1 protein.8)The full length cDNA of SpSoxE was 2843 bp encoding 491 amino acid residues,which contained a HMG-box.The results of qRT-PCR showed that SpSoxE was highly expressed in 5 pairs of appendages,7 pairs of appendages and eye-pigment formation stage1.Moreover,there were significant difference between the three periods(P<0.05).RT-PCR results demonstrated that SpSoxE was expressed only in gonads,eyestalk and brain of both sexes.qRT-PCR results indicated that SpSoxE expression in female crab brain had a significant difference compared to gonad and eyestalk(P<0.05).The expression level of SpSoxE decreased gradually at different stages of ovarian develoment,reached the lowest level in tertiary vitellogenesis(O5)and had significant difference with proliferation stage(O1)simultaneously(P<0.05).SpSoxE mRNA was expressed at the highest level in spermatid stage(T2)and was significantly different from spermatocyte stage(T1)and mature sperm stage(T3)(P <0.05).At the same time,rabbit anti-Sox9 antibody can specifically bind to proteins in the eyestalk and ovary,which demonstrated the prensent of SoxE protein.