水生生物学报2011，Vol.35Issue(2)：210-217,8.DOI:10.3724/SP.J.1035.2011.00210
中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达
CLONING AND EXPRESSION ANALYSIS OF MOLT-INHIBITING HORMONE GENE (Es-MIH) IN ERIOCHEIR SINENSIS
摘要
Abstract
Periodic molting is essential for growth and development in crustaceans. Molting is triggered by steroid hormones (ecdysteroids) which secreted by paired endocrine glands, the Y-organs. The synthesis of ecdysteroids by Yorgans is negatively regulated by a peptide neurohormone, moltinhibiting hormone (MIH), a polypeptide neurohormone released from neurosecretory cells in the X-organ/sinus gland complex of the eyestalks. To clone a full length eDNA of molt-inhibiting hormone gene from Eriocheir sinensis by RACE-PCR, degenerate primers was designed according to the partial amino acid sequences of MIH which was isolated by our lab. A novel MIH (Es-MIH, GenBank accession No. DQ341280) of 1457 bp was successfully cloned from Chinese mitten crab. It was consisted of a 330bp open reading frame, the untranslation region of 5' and 3' end were 189 and 938 nucleotides, respectively. Deduced protein contained 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-C53). A typical Crust_neurohorm domain (position 2-74 nt in mature peptide) (E-value=2.80e-33) was identified by SMART (Simple Modular Architecture Research 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 also shared high identities with Carcinus maenas (66％) and Portunus trituberculatus (62％), moreover, it showed highly identity 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 of Es-MIH were only found in eyestalks, no bands could be observed in heart, muscle, ventral nerve cord, brain and haemocytes lanes. Semi-quantitative RT-PCR gave similar results. It indicated that Es-MIH was specifically expressed in eyestalk. The recombinant Es-MIH (rEs-MIH) was expressed by pCR(g)T7/NT TOPO(g)TA expression system. The optimal time for isopropyl β-D-thiogalactopyranoside induction was 5 hours. After collection and iyses of host E. coil,rEs-MIH was purified by immobilized metal affinity chromatography column. The yield ofrEs-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 corresponding sequence of Eriocheir sinensis MIH1 (GenBank accession No. GI34597340). Low content and difficulties of purification of polypeptide neurohormone in crustacean was solved in this research by means of genetic engineering,which could facilitate understanding the probable role of MIH in development process of crab and would be helpful to crustacean culture.关键词
中华绒螯蟹/蜕皮抑制激素基因/组织表达/重组表达/纯化分类
生物科学引用本文复制引用
孙妍,张亦陈,刘逸尘,王雪惠,王宇凡,耿绪云,孙金生,杨卫军..中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达[J].水生生物学报,2011,35(2):210-217,8.基金项目
国家自然科学基金(30571421、30871907) （30571421、30871907）
天津市应用基础及前沿技术研究项目(10JCZDJC18200、09JCYBJC15000、10JCYBJC09200) （10JCZDJC18200、09JCYBJC15000、10JCYBJC09200）
国家科技支撑计划项目(2006BAD09A11) （2006BAD09A11）
天津市高等学校科技发展基金计划项目(20080618) （20080618）
天津师范大学博士基金项目(52LX18119)资助 （52LX18119）
