| Chinese mitten crab (Eriocheir sinensis) is a main kind of health aquatic products which is delicious and nutritious in China.However, sexual premature in larvae occurred widespreadly during breeding cultivate process and affected much in the yield. Research showed that gonad-inhibiting hormone was important on the regulation of the gonadal maturation.Using molecular biology methods to research gonad-inhibiting hormone, there were some gonad-inhibiting hormonegene sequence have been reported in shrimp, but not in crabs.In this paper,RACE technique was used for getting the whole sequence of GIH of E. sinensis,and the gene fragment which code the GIH was expressed.Pairs of primers were designed according as known conservative and codon usage of GIH gene sequences. Then eyestalk total RNA was extracted by using the RNAiso? Plus reagent,used RACE for amplificating GIH gene sequences. Connected gel extracted RCR product with pMD19-T, tranceformed E. coli. DH5α, screened the positive cloning and sequenced.At last,a 1458bp GIH sequence form the eyestalk of E. sinensis was obtained. The sequence has relatively high similarity with five kinds of GIH which has been known,and the similar region is in the GIH open reading frames.According to the GIH sequence of E. sinensis, prokaryotic expression primers were designed and recombinant plasmid(pET-GIH) was constructed. Used expression strains E. coli. Rosetta,a protein was successfully expressed. 15% SDS-PAGE electrophoresis showed that the recombinant protein was 10 ku and accorded with known reports.So the sequence was presumed to Chinese mitten crab GIH gene preliminary.Pured with QIAexpress purification system,the concentration of recombinant protein was 6.83μg/mL by Coomassie brilliant blue methods.This paper studied the effect of recombinant protein GIH on the ovarian development of the E. sinensis in vitro and in vivo.The result showed that recombinant protein GIH could inhibit the ovarian development. |
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