Gene Cloning、Pression Analysis And Function Research Of The Putative Molt Inhibiting Hormone(MIH1) From The Shrimp Fenneropenaeus Merguiensis

The eyestalk neuroendocrine regulation center,X-organ-sinus gland(XO-SG)synthesises and secretes the molting inhibiting hormone from Fenneropenaeus merguiensis,a neuropeptide member of the CHH family.Molting inhibiting hormone(MIH)regulates the molting process in crustaceans with molting hormone(MH)in mutual antagonistic manner,besides playing an important role in a series of activities such as growth,reproduction and osmotic adjustment.In recent years,with the development of the genetic breeding work,it is necessary to study on cloning,expression and function of gene related to growth,reproduction and molting.In Fenneropenaeus merguiensis,the cDNA、gDNA full length sequence of MIH1 gene was obtained by using RACE,RT-PCR and characterized.The expression of MIH1 gene in different tissues,different molt stage was tested by real-time PCR.At the same time,MIH1 protein was successfully induced by prokaryotic recombinant expression technology.Respectively injection of FmMIH1 recombinant proteins and double chain verificated function for FmMIH1.FmMIH1 possesses most of the characteristics of the eyestalk type-II CHH/MIH1/GIH family neuropeptide.The open reading frame of FmMIH1 consists of 318 bp encoding for a protein of 105 amino acid residues.The mature peptide of FmMIH1 consists of 76 amino acid residues,a glycine residue at position 11 of the mature peptide and 6 cysteine residues located in the conserved position of the mature peptide.The FmMIH1 gene is composed of 2 introns and 3 exons.The homology analysis showed that FmMIH1 is very more conservative in the functional structure domain,which share the highest homologies with Penaeus monodon MIH1,Litopenaeus vannamei MIH1(99%、98%).Phylogenetic analysis showed that the Fm MIH1 can cluster together with crustacean macrura,in addition that have the closest relationship with Penaeus monodon MIH1,Litopenaeus vannamei MIH1.In addition to eyestalk,high levels of Fm MIH1 transcript could also be detected in the intestine.FmMIH1 transcript level is low throughout the post-molt,early to mid-intermolt and premolt.However,a sharp increase could be observed in late intermolt(C3).For functional assay,RNA interference results show that a significant 2.7 days(P<0.05)reduction in molt cycle duration could be observed in shrimp receiving dsFmMIH1 injection.Surprisingly,injection of recombinant FmMIH1 could also cause a significant reduction of the molt cycle duration(average 1.8 days,P<0.05).We hypothesize that the recombinant protein is biological inactive but it competes with the endogenous MIH1 for carrier protein binding and consequently reduces the amount of biological MIH1 that could reach the targets.In conclusion,the result of this study will provide us new insight to manipulate molting/growth in crustaceans.