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: 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。Abstract: 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
-
Keywords:
- Eriocheir sinensis /
- Molt-inhibiting hormone /
- Cloning /
- Expression profile /
- Prokaryotic expression
-
-
[1] Cooke I M, Sullivan R E. Hormone and secretion. In: Bliss DE (Eds.), The Biology of Crustacean [M]. New York: AcademicPress. 1982, 3: 205—290
[2] Keller R. Crustacean neuropeptides: structure, function andcomparative aspects [J]. Experientia, 1992, 48(5): 439—448
[3] Van Herp F. Molecular, cyclogical, and physiological aspectsof the crutacean hyperglycemic hormones family [M]. RecentAdvances in Arthropd Endocrinology, 1998, 53—70
[4] Webster S G. Amino acid sequence of putative molt inhibitinghormone from the crab, Carcinus maenas [J]. Proceedingsof the Royal Society B-Biological sciences. B, 1991,244(1311): 247—252
[5] Lu W, Wainwright G, Olohan L A, et al. Characterization ofcDNA encoding molt-inhibiting hormone of the crab, Cancerspagurus, expression of MIH in non-X-Organ tissue [J].Gene, 2001, 278(1-2): 149—159
[6] Gu P L, Chan S M. Cloning of a cDNA encoding a putativemolting inhibiting hormone from the eyestalk of the sandshrimp, Metapenaeus ensis [J]. Molecular Marine Biologyand Biotechnology, 1998, 7(3): 214—220
[7] Sun P S. Molecular cloning and sequence analysis of acDNA encoding a molt-inhibiting hormone-like neuropeptidefrom the white shrimp Penaeus vannamei [J]. MolecularMarine Biology and Biotechnology, 1994, 3(1): 1—6
[8] Lee K J. Molecular cloning of a cDNA encoding putativemolt-inhibiting hormone from the blue crab, Callinectessapidus [J]. Biochemical and Biophysical Research Communications,1995, 209(3): 1126—1131
[9] Ohira T, Watanabe T, Nagasawa H, et al. Molecular cloningof a molt-inhibiting hormone cDNA from the kuruma prawnPenaeus japonicus [J]. Zoological Science, 1997, 14(5):785—789
[10] Sun J S, Gao C L, Xiang J H. Research on the Glutamategated channels in the neurosecretory cells of MTXO in theeyestalk of Eriocheir sinensis [J]. Acta Hydrobio1ogicaSinica, 2004, 28(6): 647—652 [孙金生, 高春蕾, 相建海.河蟹眼柄神经内分泌细胞Glu 受体研究. 水生生物学报,2004, 28(6): 647—652] Sun J S, Gao C L, Xiang J H. Research on the Glutamategated channels in the neurosecretory cells of MTXO in theeyestalk of Eriocheir sinensis [J]. Acta Hydrobio1ogicaSinica, 2004, 28(6): 647—652 [孙金生, 高春蕾, 相建海.河蟹眼柄神经内分泌细胞Glu 受体研究. 水生生物学报,2004, 28(6): 647—652]
[11] Wang X H, Sun J S, Yang W J. Isolation and activity of acrustacean hyperglycemic hormone (CHH) from Eriocheirsinensis [J]. Journal of Fisheries of China, 2006, 30(2): 150—155 [王雪惠, 孙金生, 杨卫军. 中华绒螯蟹眼柄神经多肽激素(CHH)的分离纯化及活性鉴定. 水产学报, 2006,30(2): 150—155] Wang X H, Sun J S, Yang W J. Isolation and activity of acrustacean hyperglycemic hormone (CHH) from Eriocheirsinensis [J]. Journal of Fisheries of China, 2006, 30(2): 150—155 [王雪惠, 孙金生, 杨卫军. 中华绒螯蟹眼柄神经多肽激素(CHH)的分离纯化及活性鉴定. 水产学报, 2006,30(2): 150—155]
[12] Sun J S, Liu A X, Du Y Z. Micrcostructure and ultrastructureof sinus gland in the eyestalk of Eriocheir sinensis [J]. ActaZoologica Sinica, 2001, 47(1): 27—31 [孙金生, 刘安西, 杜育哲. 中华绒螯蟹窦腺的显微和超微结构. 动物学报,2001, 47 (1): 27—31] Sun J S, Liu A X, Du Y Z. Micrcostructure and ultrastructureof sinus gland in the eyestalk of Eriocheir sinensis [J]. ActaZoologica Sinica, 2001, 47(1): 27—31 [孙金生, 刘安西, 杜育哲. 中华绒螯蟹窦腺的显微和超微结构. 动物学报,2001, 47 (1): 27—31]
[13] Shangguan B M, Li S J. Cytologic research of X-organ inScylla serrata [J]. Acta Oceanologica Sinica, 1994, 16(6):116—121 [上官步敏, 李少菁. 锯缘青蟹X 器官神经分泌细胞的细胞学研究. 海洋学报, 1994, 16(6): 116—121] Shangguan B M, Li S J. Cytologic research of X-organ inScylla serrata [J]. Acta Oceanologica Sinica, 1994, 16(6):116—121 [上官步敏, 李少菁. 锯缘青蟹X 器官神经分泌细胞的细胞学研究. 海洋学报, 1994, 16(6): 116—121]
[14] Shangguan B M, Li S Q. Micrcostructure and ultrastructureof sinus gland in the eyestalk of Scylla serrata [J]. Acta ZoologicaSinica, 1995, 41(4): 341—346 [上官步敏, 李少菁.锯缘青蟹窦腺显微和超微结构研究. 动物学报, 1995,41(4): 341—346] Shangguan B M, Li S Q. Micrcostructure and ultrastructureof sinus gland in the eyestalk of Scylla serrata [J]. Acta ZoologicaSinica, 1995, 41(4): 341—346 [上官步敏, 李少菁.锯缘青蟹窦腺显微和超微结构研究. 动物学报, 1995,41(4): 341—346]
[15] Kang X J, Li Y. Neuroscretory structure of eyestalk inFennropenaeus chinensis [J]. Journal of Hebei University(Natural Science Edition), 1998, 18(1): 45—48 [康现江, 李阳. 中国对虾眼柄的神经分泌结构. 河北大学学报(自然科学版), 1998, 18(1): 45—48] Kang X J, Li Y. Neuroscretory structure of eyestalk inFennropenaeus chinensis [J]. Journal of Hebei University(Natural Science Edition), 1998, 18(1): 45—48 [康现江, 李阳. 中国对虾眼柄的神经分泌结构. 河北大学学报(自然科学版), 1998, 18(1): 45—48]
[16] Yao B. Ultrastructure of neurosecretory cell in eyestalk ofMacrobrachium rosenbergii [J]. Supplement to the Journalof Sun Yatsen University, 1995, (3): 64—67 [姚泊. 罗氏沼虾眼柄神经分泌细胞的超微结构. 中山大学学报论丛,1995, (3): 64—67] Yao B. Ultrastructure of neurosecretory cell in eyestalk ofMacrobrachium rosenbergii [J]. Supplement to the Journalof Sun Yatsen University, 1995, (3): 64—67 [姚泊. 罗氏沼虾眼柄神经分泌细胞的超微结构. 中山大学学报论丛,1995, (3): 64—67]
[17] Wang Z Z. Molecular cloning and sequence analysis of fulllength cDNA encoding molt-inhibiting hormone fromFennropenaeus chinensis [J]. Acta Genetica Sinica, 2003,30(2): 128—134 [王在照. 中国对虾蜕皮抑制激素全长cDNA 的克隆和序列分析. 遗传学报, 2003, 30(2): 128—134] Wang Z Z. Molecular cloning and sequence analysis of fulllength cDNA encoding molt-inhibiting hormone fromFennropenaeus chinensis [J]. Acta Genetica Sinica, 2003,30(2): 128—134 [王在照. 中国对虾蜕皮抑制激素全长cDNA 的克隆和序列分析. 遗传学报, 2003, 30(2): 128—134]
[18] Song X, Zhou K Y, Ma C Y. Molecular cloning and Northernblot analysis of a cDNA fragment of molt-inhibiting hormonegene 1 from Eriocheir sinensis [J]. Journal of FisherySciences of China, 2003, 10(5): 353—358 [宋霞, 周开亚,马长艳. 中华绒鳌蟹蜕皮抑制激素l(MIH1)基因的cDNA片段克隆和Northern 印迹分析. 中国水产科学, 2003,10(5): 353—358] Song X, Zhou K Y, Ma C Y. Molecular cloning and Northernblot analysis of a cDNA fragment of molt-inhibiting hormonegene 1 from Eriocheir sinensis [J]. Journal of FisherySciences of China, 2003, 10(5): 353—358 [宋霞, 周开亚,马长艳. 中华绒鳌蟹蜕皮抑制激素l(MIH1)基因的cDNA片段克隆和Northern 印迹分析. 中国水产科学, 2003,10(5): 353—358]
[19] Lee K J, Kim H W, Gomez A M, et al. Molt-inhibiting hormonefrom the tropical land crab Gecarcinus lateralis: cloning,tissue expression, and expression of biologically activerecombinant peptide in yeast [J]. General and ComparativeEndocrinology, 2007, 150(3): 505—513
[20] Chen H Y, Watson R D, Chen J C. Molecular characterizationand gene expression pattern of two putative molt- inhibitinghormones from Litopenaeus vannamei [J]. General andComparative Endocrinology, 2007, 151(1): 72—81
[21] Chan S M. PCR cloning and expression of the molt - inhibitinghormone gene for the crab Charybdis feriatus [J]. Gene,1998, 224(1-2): 23—33
[22] Chang E S, Keller R. Determination of the amino acid sequenceof the molt - inhibiting hormone from the edible crab,Cancer pagurus [J]. Neuropeptides, 1996, 30(1): 95—101
[23] Klein J M, Mangerich S, Kleijn D P, et al. Molecular cloningof crustacean putative molt-inhibiting hormone (MIH) precursor[J]. FEBS Letters, 1993, 334 (1): 139—142
[24] Nagasawa H. Isolation and amino acid sequence of amolt-inhibiting hormone from the American crayfish, Procambarusclarkii [J]. Bioscience Biotechnology and Biochemisty,1996, 60(3): 554—556
[25] Katayama H, Ohira T, Nagata S, et al. Structure-activityrelationship of crustacean molt-inhibiting hormone from theKuruma Prawn Marsupenaeus japonicus [J]. Biochemistry,2004, 43(30): 9629—9635
[26] Qiu G F, Zhang A P, Lou Y D. cDNA cloning and expressionanalysis of molt-inhibiting hormone in the mud crab Scyllaserrata [J]. Journal of Fisheries of China, 2003, 27(3): 209—212 [邱高峰, 张爱萍, 楼允东. 锯缘青蟹蜕皮抑制激素cDNA 的分子克隆及其表达分析. 水产学报, 2003, 27(3):209—212] Qiu G F, Zhang A P, Lou Y D. cDNA cloning and expressionanalysis of molt-inhibiting hormone in the mud crab Scyllaserrata [J]. Journal of Fisheries of China, 2003, 27(3): 209—212 [邱高峰, 张爱萍, 楼允东. 锯缘青蟹蜕皮抑制激素cDNA 的分子克隆及其表达分析. 水产学报, 2003, 27(3):209—212]
计量
- 文章访问数:
- HTML全文浏览量: 0
- PDF下载量:
下载: