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

作　　者：孙妍[1,2] 张亦陈[1] 刘逸尘[1] 王雪惠[2] 王宇凡[1] 耿绪云[2] 孙金生[1,2] 杨卫军[3] 

机构地区：[1]天津师范大学生命科学学院,天津300387 [2]天津市水产养殖病害防治中心,天津300221 [3]浙江大学生命科学学院,杭州310058

出　　处：《水生生物学报》2011年第2期210-217,共8页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(30571421;30871907);天津市应用基础及前沿技术研究项目(10JCZDJC18200;09JCYBJC15000;10JCYBJC09200);国家科技支撑计划项目(2006BAD09A11);天津市高等学校科技发展基金计划项目(20080618);天津师范大学博士基金项目(52LX18119)资助

摘　　要：根据实验室分离自中华绒螯蟹(Eriocheir sinensis)的一种蜕皮抑制激素(Molting-inhibiting hormone,MIH)N端氨基酸测序结果设计简并引物,采用RACE方法,首次从中华绒螯蟹眼柄中克隆到蜕皮抑制激素基因全长cDNA(Es-MIH,GenBank登录号:DQ341280),该基因全长为1457 bp,开放阅读框为330 bp,编码110个氨基酸(含有35个氨基酸的信号肽);其成熟肽包含C7-C44、C24-C40和C27-C53三个二硫键,有典型的CHH家族结构域。该cDNA编码的氨基酸序列与地蟹(Gecarcinus lateralis)MIH同源性最高,达到了85%。Northern杂交和半定量RT-PCR显示蜕皮间期成体蟹仅在眼柄中有MIH基因表达,提示该基因的表达具有一定组织特异性。利用pCR T7/NT TOPO TA系统重组表达MIH成熟肽,纯化的重组蛋白得率为0.3 g/L,纯化产物经质谱鉴定为中华绒螯蟹MIH。研究解决了CHH家族神经肽在机体中的表达量少,直接纯化较难的问题,为深入研究MIH的作用机制和在生产上控制中华绒螯蟹蜕皮和生长奠定了基础。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 neurohor-mone released from neurosecretory cells in the X-organ/sinus gland complex of the eyestalks.To clone a full length cDNA of molt-inhibiting hormone gene from Eriocheir sinensis by RACE-PCR,degenerate primers was designed ac-cording 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 pro-tein contained a putative signal peptide of 35 amino acids and a mature peptide of 75 amino acids.Es-MIH contains 6 conserved cysteines which formed three disulfide bonds（C7-C44,C24-C40 and C27-C53）.A typical Crust_neurohorm do-main（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 eyesta

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 号：Q781[生物学—分子生物学] Q786