| Insulin-like androgenic gland hormone(IAG)is secreted by the specific androgenic gland(AG)of crustaceans.It plays an important role in gender differentiation and the maintenance of male secondary sex characteristics.However,the molecular mechanism of sex differentiation in Fenneropenaeus merguiensis is still unknown.Therefore,the studying of molecular characteristics of sex differentiation related genes can provide a theoretical basis for elucidating the mechanism of sex differentiation in F.merguiensis.In this study,RT-PCR and RACE techniques were used to clone the full length of c DNA,genomic DNA and 5’ flanking region of IAG gene in F.merguiensis,and bioinformatics analysis was performed.The expression characteristics of Fm IAG genes in different tissues,different molting and larval stages were analyzed by real-time fluorescence quantitative PCR(q RT-PCR)technique.The relationship between Fm IAG gene and other gender related genes was analyzed by RNA interference(RNAi)and q RT-PCR technology.In addition,the relationship between the Fm IAG gene and the eyestalk ganglia was preliminarily studied by q RT-PCR technology.The full length of c DNA sequence of Fm IAG was 2092 bp with an ORF of 510 bp,which encoded 169 amino acids pre-propeptide.The IAG polypeptide of F.merguiensis is made up of a signal peptide(34 aa)and a mature peptide(135 aa).The mature peptide is composed of B chain,C peptide and A chain.The cleavage site of B chain and C peptide is RVSR,C peptide and A chain cleavage site RVKR.Similar to IAG of other crustaceans,the six Cystine(Cys)residues in Fm IAG mature peptide are highly conserved.The predicted N terminal glycosylation site Asn37 is located in the B chain.The amino acid seqence of Fm IAG gene is most similar to that of decapod IAG genes.The Fm IAG evolution tree results showed that Fm IAG and the IAGs of other prawns were clustered in one branch,and the homology of the IAG genes of Fenneropenaeus chinensis,Penaeus monodon and Litopenaeus vannamei was the highest showing,91%,81% and 80% identity respectively.However,the gene structure characteristics of IAG are different from those of other crustaceans.Fm IAG gene has 3 exons and 2 introns.The 5’ flanking sequence of the Fm IAG gene is highly homologous to the IAG gene 5’ flanking sequence of F.chinensiss(>80%).The result suggest that they shared several identical potential transcription factor binding sites: TBP,Fox F1,Sp1,Ste11+,AR and TEF-1.Results from q RT-PCR showed that Fm IAG was expressed exclusively in AG of F.merguiensi.During the molting cycle,the expression of Fm IAG gene in the late intermolt was low until after proecdysis and before postecdysis,and the transcription level in the early intermolt was reduced to the lowest level.Fm IAG gene transcript can be detected in larval development stage,with the highest transcript level detected in the nauplii stage.Knock-down of Fm IAG transcript by ds RNA showed that the expression level of Fm IAG decreased to the lowest level in 72 h.At 24 h after injection of Fm IAG-ds RNA,the expression of vitellogenin genes Vg1 and Vg2 appeared in testis and hepatopancreas,respectively.The expression of Fm VASA gene in the testis decreased slightly.Interestingly,the expression of a Fm CHH-like gene in the AG decreased to the lowest level at 72 h,and the pattern of variation of this Fm CHH-like gene was similar to that of the Fm IAG gene.It is possible that the Fm CHH-like gene may be involved in the regulation of the expression of the Fm IAG gene.q RT-PCR shows that unilateral eyestalk extirpation experiments and in vitro incubation experiments show that the secretory substance of the eyestalk can directly act on Fm IAG and negatively regulates it.To summarize,results from molecular characteristization of Fm IAG gene in F.merguiensis had provided an in-depth understanding for the mechanism of male sex control shrimp. |
