CLONING AND EXPRESSION ANALYSIS OF MOLT-INHIBITING HORMONE GENE|(Es-MIH) IN ERIOCHEIR SINENSIS

Periodic molting is essential for growth and development in crustaceans. Molting is triggered by steroidhormones (ecdysteroids) which secreted by paired endocrine glands, the Y-organs. The synthesis of ecdysteroids byYorgans is negatively regulated by a peptide neurohormone, moltinhibiting hormone (MIH), a polypeptide neurohormonereleased from neurosecretory cells in the X-organ/sinus gland complex of the eyestalks. To clone a full lengthcDNA of molt-inhibiting hormone gene from Eriocheir sinensis by RACE-PCR, degenerate primers was designed accordingto the partial amino acid sequences of MIH which was isolated by our lab. A novel MIH (Es-MIH, GenBankaccession No. DQ341280) of 1457 bp was successfully cloned from Chinese mitten crab. It was consisted of a 330bpopen reading frame, the untranslation region of 5′and 3′end were 189 and 938 nucleotides, respectively. Deduced proteincontained a putative signal peptide of 35 amino acids and a mature peptide of 75 amino acids. Es-MIH contains 6conserved cysteines which formed three disulfide bonds (C7-C44, C24-C40 and C27-C53C7-C44、C24-C40 and C27-C53). A typical Crust_neurohorm domain(position 2—74 nt in mature peptide) (E-value=2.80e-33) was identified by SMART (Simple Modular ArchitectureResearch Tool) in Expasy. There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.Multiple alignment results showed that Es-MIH has the highest identity with Gecarcinus lateralis MIH (85%), it alsoshared high identities with Carcinus maenas (66%) and Portunus trituberculatus (62%), moreover, it showed highlyidentity with MIH from shrimps, such as Metapenaeus ensis MIH (44%), Fenneropenaeus chinensis MIH (43%),Penaeus monodon MIH (43%) and Litopenaeus vannamei MIH (42%). Northern blotting reveled that transcripts ofEs-MIH were only found in eyestalks, no bands could be observed in heart, muscle, ventral nerve cord, brain andhaemocytes lanes. Semi-quantitative RT-PCR gave similar results. It indicated that Es-MIH was specifically expressedin eyestalk. The recombinant Es-MIH (rEs-MIH) was expressed by pCR?T7/NT TOPO?TA expression system. The optimaltime for isopropyl β-D-thiogalactopyranoside induction was 5 hours. After collection and lyses of host E. coli,rEs-MIH was purified by immobilized metal affinity chromatography column. The yield of rEs-MIH could reach 0.3 g/L.The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the correspondingsequence of Eriocheir sinensis MIH1 (GenBank accession No. GI34597340). Low content and difficulties ofpurification of polypeptide neurohormone in crustacean was solved in this research by means of genetic engineering,which could facilitat