中华绒螯蟹性腺抑制激素基因克隆与序列分析
摘要
中华绒螯蟹(Eriocheir sinensis)是我国名优淡水养殖业的主要品种,也是我国一种重要的经济蟹类,广泛存在于我国南北各地淡水水域中。而普遍发生1龄蟹性早熟的现象严重影响其产量,给生产带来巨大损失。
本文用基因工程方法来研究在中华绒螯蟹性腺发育过程起重要作用的性腺抑制激素。主要分为两部分：(1)获得中华绒螯蟹性腺抑制激素编码框内部分序列片段。(2)通过3’RACE得到基因序列片段的3’端。
本实验在解剖中华绒螯蟹视上神经节的基础上,使用TRIzol试剂提取中华绒螯蟹视上神经节总RNA, RT-PCR扩增编码性腺抑制激素(GIH)基因片段。用改进的冻融离心法纯化片段,片段与pMD19-T载体连接,转化大肠杆菌JM109,经抗性筛选、PCR鉴定、质粒电泳鉴定等方法筛选阳性克隆并测序,所测得100bp序列与三种已知性腺抑制激素前体序列相似性较高,其中有两段序列共40bp与三种性腺抑制激素一致,且相似区域都在三种性腺抑制激素的编码框架之内。
用所测序列中与三种龙虾性腺抑制激素前体相似性最高的两个区域设计特异引物,再次提取中华绒螯蟹视上神经节总RNA，RT-PCR扩增到5个片段。分别克隆后,其中四个序列在5’端与上述序列有很高的相似性,仅有2个碱基的差别。其中一个序列含有polyA尾,通过序列分析发现测序结果与三种龙虾性腺抑制激素前体有较高的相似性,推测所得序列为中华绒螯蟹性腺抑制激素基因片段。
Chinese mitten crab (Eriocheir sinensis) is an important economic crab, and one of the famous freshwater species for aquaculture. It exists throughout South and North of freshwater in China. But sexual premature in larvae occurred widespreadly and affected the yield badly.
In this paper, genetic engineering technique was used for studying on gonad-inhibiting hormone(GIH), which has an important effect on chinese mitten crab gonad growth. It falls into two parts:(1) obtain a part of open reading frames of chinese mitten crab's GIH sequence. (2) obtain the 3'end after 3'RACE.
During the experiment, optic ganglion of chinese mitten crab was dissected firstly. Then total RNA was extracted by using the TRIzol reagent. A part of GIH cDNA fragments in open reading frames was obtained after RT-PCR. The DNA fragments was purified by the modified freezing-thawing method. Then the DNA fragments was jointed with pMD19-T vector. Recombinant plasmid was transformed to JM109. The confirmation was gained from antibiotic resistance, PCR identification and agarose gel electrophoresis of plasmid. After DNA sequencing, 100bp sequence has relatively high similarity with three kinds of GIH which has been known. The similar region is in the GIH open reading frames.
The Primers which were used for 3'RACE were designed in the similar region. Then total RNA was extracted again. Five DNA fragments was obtained after RT-PCR. They were cloned respectively. After DNA sequencing, the 5'end has relatively high similarity in four DNA fragments,just 2bp dissimilarity. One of the DNA fragments has polyA in the 3'end. The sequencing result has relatively high similarity with three kinds of GIH which has been known. The DNA fragments was presumed to a part of chinese mitten crab GIH gene.
本文用基因工程方法来研究在中华绒螯蟹性腺发育过程起重要作用的性腺抑制激素。主要分为两部分：(1)获得中华绒螯蟹性腺抑制激素编码框内部分序列片段。(2)通过3’RACE得到基因序列片段的3’端。
本实验在解剖中华绒螯蟹视上神经节的基础上,使用TRIzol试剂提取中华绒螯蟹视上神经节总RNA, RT-PCR扩增编码性腺抑制激素(GIH)基因片段。用改进的冻融离心法纯化片段,片段与pMD19-T载体连接,转化大肠杆菌JM109,经抗性筛选、PCR鉴定、质粒电泳鉴定等方法筛选阳性克隆并测序,所测得100bp序列与三种已知性腺抑制激素前体序列相似性较高,其中有两段序列共40bp与三种性腺抑制激素一致,且相似区域都在三种性腺抑制激素的编码框架之内。
用所测序列中与三种龙虾性腺抑制激素前体相似性最高的两个区域设计特异引物,再次提取中华绒螯蟹视上神经节总RNA，RT-PCR扩增到5个片段。分别克隆后,其中四个序列在5’端与上述序列有很高的相似性,仅有2个碱基的差别。其中一个序列含有polyA尾,通过序列分析发现测序结果与三种龙虾性腺抑制激素前体有较高的相似性,推测所得序列为中华绒螯蟹性腺抑制激素基因片段。
Chinese mitten crab (Eriocheir sinensis) is an important economic crab, and one of the famous freshwater species for aquaculture. It exists throughout South and North of freshwater in China. But sexual premature in larvae occurred widespreadly and affected the yield badly.
In this paper, genetic engineering technique was used for studying on gonad-inhibiting hormone(GIH), which has an important effect on chinese mitten crab gonad growth. It falls into two parts:(1) obtain a part of open reading frames of chinese mitten crab's GIH sequence. (2) obtain the 3'end after 3'RACE.
During the experiment, optic ganglion of chinese mitten crab was dissected firstly. Then total RNA was extracted by using the TRIzol reagent. A part of GIH cDNA fragments in open reading frames was obtained after RT-PCR. The DNA fragments was purified by the modified freezing-thawing method. Then the DNA fragments was jointed with pMD19-T vector. Recombinant plasmid was transformed to JM109. The confirmation was gained from antibiotic resistance, PCR identification and agarose gel electrophoresis of plasmid. After DNA sequencing, 100bp sequence has relatively high similarity with three kinds of GIH which has been known. The similar region is in the GIH open reading frames.
The Primers which were used for 3'RACE were designed in the similar region. Then total RNA was extracted again. Five DNA fragments was obtained after RT-PCR. They were cloned respectively. After DNA sequencing, the 5'end has relatively high similarity in four DNA fragments,just 2bp dissimilarity. One of the DNA fragments has polyA in the 3'end. The sequencing result has relatively high similarity with three kinds of GIH which has been known. The DNA fragments was presumed to a part of chinese mitten crab GIH gene.
引文
[1]王鹏飞,月芬.河蟹扣蟹养殖过程中出现性早熟的原因及防范措施.水产科学,1994(4)：29-30.
[2]金刚.早熟河蟹的养殖生态学及渔业价值评估.水生生物学报,1998,22(2)：143-147.
[3]杨洪良.稻田养扣蟹性早熟原因及对策.中国水产,1994,(11)：24-25.
[4]柳志会,孙福新.中华绒螯蟹池塘养殖性早熟产生原因的初步分析与其预防.海水养殖1992,(1-2)：66-69.
[5]陈贤龙,沈江平.一龄河蟹性早熟现象观察.水产科技情报,1999,26(1)28-31.
[6]何正侃,印骏,朱雅珠.密度、营养与河蟹蟹种生长及性早熟之间的关系.水产科技情报,1999,26(2)：73-76.
[7]徐如卫,江锦坡,陆开宏等.河蟹性早熟原因的初步研究.浙江海洋学院学报(自然科学版),2001,20(3)：196-198.
[8]丛宁,臧素娟,凌爱珍等.河蟹研究的若干进展.内陆水产,1994,11：2-5.
[9]王勇军.周期性温度变化对幼蟹生长发育的影响.水产养殖,1999,4：11-20.
[10]赵晓红,送笛.温度、降盐度及pH对中华绒螯蟹幼体变态的影响.大连水产学院学报2001,16(4)：249-256.
[11]吴琅虎,李杰,温周瑞等.河蟹性早熟产生原因及预防措施的探讨.内陆水产,1999,6：15-18.
[12]胡兆群.控制河蟹性早熟技术研究.河北渔业,1998,5：9-11.
[13]朱雅珠,张根玉,王建军等.饲料中适宜的动植物蛋白比与河蟹蟹种生长及性早熟的相关.水产科技情报,1999,26(1)：59-63.
[14]李云峰,康现江,赵晓瑜等.河蟹性早熟发生的相关内在因素研究进展.水产科学,2005,24(2)：34-36.
[15]周开亚,高志千.RAPD标记鉴别中华绒螯蟹种群初步研究.应用与环境生物学报,1999,5(2)：176-180.
[16]张德隆,杜晓燕,赵金利等.河蟹性早熟成因及控制方法研究.淡水渔业,2001,31(4)：36-39.
[17]羊茜,占家智.辽河蟹与长江蟹生长速度的对比试验.淡水渔业,1999,29(10)：40-42.
[18]孙金生,刘安西,杜育哲等.中华绒螯蟹窦腺的显微和超微结构.动物学报,2001,47(1)：27-31.
[19]赵维信,陆剑峰.中华绒螯蟹大颚器官激素生物合成与性早熟的关系.水产学报,2003,27(4)： 289-294.
[20]赵维信,陆剑峰.中华绒螯蟹Y-器官在蜕皮周期中的超微结构.中国水产科学,2004,11(1)：74-76.
[21]吴嘉敏,姜新耀.中华绒螯蟹血淋巴钙离子和17p-雌二醇浓度与性早熟的关.水产学报,2001,25(2)：112-115.
[22]成永,旭王武.虾蟹类脂类营养与中华绒鳌蟹性早熟.科学养鱼,2000,6：39-40.
[23]吴嘉敏,姜新耀.中华绒螯蟹血淋巴和肝胰腺的总蛋白含量与性早熟的关系.水产学报,2000,24(4)：306-311.
[24]陈再忠,成勇旭,王武.早熟期间中华绒螯蟹肝胰腺指数、肝脂含量及脂肪酸组成的变.水产学报,2003,27(1)：57-61.
[25]康现江,米娅,孙辉建等.中华绒螯蟹眼柄神经内分泌系统结构的研究.华东师范大学学报(自然科学版),1998：22-26.
[26]康现江,郭明申,米娅等.中华绒螯蟹眼柄神经分泌系统结构的研究：Ⅲ.窦腺的显微和超微结构.中国动物科学研究,1994：103-106.
[27]赵维信,贾江,安苗.外源激素和眼柄提取物对罗氏沼虾卵目细胞的离体诱导作用.上海水产大学学报,1996,5(4)：221-225.
[28]堵南山,赖伟,安婴等.中华绒螯蟹受精的细胞学研究.中国科学(B辑),1992,36(3)：260-265.
[29]王在照,相建海.甲壳动物CHH家族神经激素结构和功能研究进展.水产学报,2001,25(2)：175-180.
[30]Kleijn P.V. De and Franqois V. H. Molecular biology of neurohormone precursors in the eyestalk of Crustacea. Comp. Biochem. Physiol,1995,112 (4):573-579.
[31]Gu P.L, Chan S. M. Cloning for a cDNA encoding a putative Molt-Inhibiting Hormone from the eyestalk of the sand shrimp Metapenaeus ensis. Molecular Marine Biology and Biotechnology,1998, 7(3):214-220.
[32]Soyez D, LeCaer J. P, Noel P Primary structure of two isoforms of the vitellogenesis-inhibiting hormone from the lobster Homarus americanus. Neuropeptides 1991.20,25-32.
[33]Aguilar, M. B, Quackenbush, L. S, Hunt, D. T, et al. Identification, purification and initial characterization of the vitellogenesis-inhibiting hormone from the Mexican crayfish Procambarus bouOieri Ortmann. Comp. Biochem. Physiol.1992.102B,491-498.
[34]Keller R. Crastacean neuropeptides:structures, functions and comparative aspects, experimenti-al,1992,48(5):439-448.
[35]蔡生力.甲壳动物内分泌学研究与展望.水产学报,1998,22(2)：154-161.
[36]Kegel G, Reicwein B, Weese S, et al. Amino acid sequence of Crustacean Hyperglycemic Hormore(CHH) from the shore crab Carcinus maenas. FEBS Lett,1989,255:10-14.
[37]Tensen C. P, Keijn D. P., Van H. F. Cloning and sequence analysis of cDNAs encoding two crustacean hyperglycemic hormore in the lobster, Homarus americanus. Eur J Biochem,1991,200:103-106.
[38]Martin G, Sorokine O, Van D. A. Isolation and molecular characterization of a hyperglycemic neuropetide from the sinus gland of the terrestial isopod, Armadillidium vulgare (Crustacea). Eur J Biochem,1993,211:601-607.
[39]Kegel G, Reicwein B, Tense C. P, et al. Amino acid sequence of Crustacean Hyperglycemic Hormore(CHH) from the crayfish Orconectes limosus. Emergence of a novel neuropeptide family. Peptides,1991,12:909-913.
[40]Huberman A, Aguilar M B, Brew K, et al. Primary structure of the major isomorph of Crustacean Hyperglycemic Hormore(CHH-1) from the sinus gland of the Mexican crayfsh Procambarus bouvieri (Ortmmann):interspecies comparison. Peptides,1993,14:7-16.
[41]Yasuda A, Yasuda Y, Fujita T, et al. Characterization of crustacean hyperglycemic hormore from the crayfish (Procambarus clarkii):Multiplicity of molecular stereoversion and diverse functions. Gen Comp Endocr,1994,95:387-398.
[42]Ohira T, Watanabe T, NagasawaH, et al. Molecular cloning of cDNAs encoding four crustacean hyperglycemic hormones and a molt-inhibiting hormone from the kuruma prawn Penaeus japonicus. Proceedings of the ⅩⅢ international congress of comparative endocrinology. Yokohama,1998,83-86.
[43]Chung J. S, Wilkinson M C, Webster S G Amino acid sequences of both isoforms of crustacean hyperglycemic hormone(CHH) and corresponding precursor-related peptide in Cancer Pagurus. Regul Pept,1998,7:17-24.
[44]Gu P. L, Chan S M. The shrimp hyperglycemic hormone-like neuropeptide is encoded by multiple copies of genes arranged in a cluster. FEBS Lett,1998,441:397-403.
[45]Marco H. G, Brandt W, Gaede G. Elucidation of the Amino Acid Sequence of a Crustacean Hyperglycemic Hormone from the Spiny Lobster, Jasus lalandii Bioch BioPhys Res Comrnun, 1998,248:578-583.
[46]Sithigorngul W., Jaideechoey S., Saraithongkum W, et al. Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.J Exp Zool,1999,284:217-224.
[47]Ju B, Khoo H. W. Characterization of crustacean hyperglycemic hormone(CHH) mRNA transcripts and genomic sequences in Macrobrachium lanchesteri.1998. Submitted to the EMBL/GenBank/DDBJdatabase.
[48]Davey M. L, Hall M. R, Willis R H,et al.Five crustacean hyperglycemic family hormones of Penaeus monodon:complementary DNA sequence and identification in single sinus glands by electrospray ionization-fourier transform mass spectrometry.Mar Biotechnol,2000,2:80-91.
[49]Huberman. Secordary structure of a crustacean neuropeptide hormone family by means of CD. Peptides,1989,10:1113-1115.
[50]Mattson M. P. E. spaziani.5-Hydroxy-trytamine mediates release of molt-inhibiting hormone activity from isolated crab eyestalk ganglia.Bio Bull,1985,169:246-255.
[51]Ohira T, Watansbe T. Molecular cloning of a molt-inLhibiting hormone form the kuruma prawn Penaeus japonicus. Zoological Science,1997,14:785-789.
[52]Mark P, Mattson, Eugene, et al. Regulation of crab y-organ steroidogenesis invitro:evidence that ecdysteroid production increases through activation of cAMP-phosphdiesterase by calcium-calmodalin. Molecular and cellular Endocrinology,1986,48:135-151.
[53]Lachaise F., Roux A. Le, Hubert M. The molting gland of crustaceans:localization, activity and endocrine control. Crustacean Bio,1993,13(2):198-234.
[54]Sedlmeier. Regulation of ecdysteroid biosynihesis in crayfish y-organ.1 Role of cyclic nucleotides. Exp.2001,1993,265:448-453.
[55]Nagasawa H.Isolation and amino acid sequence of a molt-inhibiting hormone from the American crayfish, Procambarus clartii. Biosci Biotech.Biochem,1996,60:554-556.
[56]Klein J. M., Mangerich S., Kleijn D. P. V, et al. Molecular cloning of crustacean putitive molting-inhibiting hormone(MIH)Precursor. FEBS,1993,334:139-142.
[57]Sun P. S. Molecular cloning and sequence analysis of a cDNA encoding a molt-inhibiting hormone-like neuropeptide from the white shrimp Penaeus vannamei. Mol Mar Biol Biotech,1994, 3:1-6.
[58]Lee K. J. Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus.Biochem Biophys Res Commun,1995,209:1126-1131.
[59]Chung J. S., Wilkinson M. C., Webster S. G. Determination of the amino acid sequence of the molt-inhibiting hormone from the edible crab, Cancer pagurus.Neuropeptides,1996,30:95-101.
[60]Aguilar M. B., Falchetto R, ShabanowitzJ, et al. Complete primary structure of the molt-inhibiting honnone of the Mexican crafish Procambarus bouvieri. Peptide,1996,17(3):367-374.
[61]Umphrey H. R., Lee K. J., Watson R. D., et al. Molecular cloning of a cDNA encoding molt-inhibiting hormone of the crab, Cancer magister.Mol Cell Endocrinol,1998,136:145-149.
[62]王在照,相建海,崔朝霞.编码中华绒螯蟹蜕皮抑制激素基因的cDNA片段克隆和序列分析.海洋与湖沼,2002,33(4)：432-438.
[63]王在照,焦传珍,张晓军等.中国对虾蜕皮抑制激素全长cDNA的克隆及序列分析.遗传学报,2003,30(2)：128-134.
[64]王在照,相建海.编码鹰爪虾蜕皮抑制激素基因的cDNA片段的克隆和序列分析.水产学报,2002,26(6)：487-492.
[65]Meusy J. J., Martin G., Soyez D., et al. Immunochemical and immunocytochemical studies of the crustacean vitellogenesis-inhibiting hormone (VIH). Gen Comp Endocrinol,1987,67(3):333-41.
[66]Kleijn D.P., Janssen K.P.,Waddy S.L, et al. Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American Lobster Homarus americanus J Endocrinol.1998,156(2):291-298.
[67]Edomi P., Azzoni E., Mettulio R., et al. Gonad-inhibiting hormone of the Norway Lobster(Nephrops norvegicus):cDNA cloning,expression,recombinant protein production,and immunolocalization.Gene, 2002,284,93-102.
[68]顾志敏,何林岗.中华绒螯蟹发育周期的组织学细胞学观察.海洋与湖沼,1997,28(2)：138-145.
[69]陈炳良,堵南山.中华绒螯蟹幼体中肠的超微结构.海洋与湖沼,1992,23(5)：533-535.
[70]堵南山,陈炳良,赖伟.中华绒螯蟹幼体消化系统发育的研究.海洋与湖沼,1992,23(1)：79-82.
[71]顾志敏,何林岗.切除单侧眼柄对中华绒螯蟹蜕皮、生长、成熟的影响.淡水渔业,1991,5：10-13.
[72]康现江.中华绒螯蟹眼柄神经内分泌系统结构的研究：Ⅲ窦腺的显微和超微结构,中国动物科学研究,1994：103-106.
[73]康现江,米娅,刘彬彬等.摘除眼柄对中华绒螯蟹精巢发育及其氨基酸含量的影响.台湾海峡,2000,19(3)：360-364.
[74]赵维信.十足目甲壳动物生殖激素对卵巢得作用及其调控.上海水产大学学报,200110(2)：166-171.
[75]林远声,邓锋,廖家遗.罗氏沼虾眼柄性腺抑制激素(GIH)的分离纯化及性质的研究.中山大学学报(自然科学版),2000,39(2)：93-95.
[76]Adiyodi, R. G, Bliss D. E, Mantel, et al. Reproduction and its control. In The Biology of Crustacea Academic Press, New York,1985,9:147-215.
[77]Weerawan S., Sompong J, Wanpen S., et al. Purification and characterization of an isoform of Crustacean Hyperglycemic Hormone from the eyestalk of Macrobrachium rosenbergii. Journal of Experimental Zoology,1999,284:217-224.
[78]Soyez D, Deijinen J. E, Martin M. Isolation and characterization of a vitellogene-sis-inhibiting factor from sinus glands of the lobster Homarus americanus.J Exp 2001,1987,244:479-484.
[79]Soyez D, Lecaer J. P, Noel P. Y, et al. Primary structure of two isoforms of the vitellogenesis-inhibiting hormone from the lobster Homarus americanus. Neuropeptides 1991.20,25-32.
[80]Fingerman M. Inhibition of ovarian growth by cadmium in the fiddler crab, Ucapugilator (Decapoda, ocypodidae). Ecotoxicol Environ Saf.,2000,46(2):202-206.
[81]Kleijn D. P., Sleutels F. J, Martens G. J, et al. Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone(GIH) in the lobster Homarus americanus.FEBS Lett,1994, 353:255-258.
[82]Edomi P., Azzoni E., Mettulio R., et al. Gonad-inhibiting hormone of the Norway Lobster(Nephrops norvegicus):cDNA cloning,expression, recombinant protein production,and immunolocalization.Gene 2002,284,93-102.
[83]Gu S, Tobe B. K, et al. Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis Peptides,2002,23:1875-1883.
[84]Ollivaux, C., Vinh J., Soyez D, et al. Crustacean hyperglycemic hormone family peptides in the lobster Homarus gammarus:occurence of genetic variants and posttranslationally modified isoforms GenBank DQ181793.2005.
[85]张庆华,茅矛,陈竺.基因组研究中全长cDNA克隆的策略.生物工程进展,2000,20(4)：3-5.
[86]Lohey J, Ellotf J, Cwirlas J, et al. Polymerase chain reaction with single-sided specificity:analysis of T cell receptor delta chain. Science,1989,243(488):217-222.
[87]Oharao J. H, Doritrl J, Gilbert C. Direct genomic sequencing of bacterial DNA:the pyruvate kinase 1 gene of Escherichia coli. Proc Natl Acad Sci USA,1989,86(18):6883-6887.
[88]Fehrc C, Fickova M, Hiemke C, et al. Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase6:an improved method to determine 5'-and3'-cDNA ends. Brain Res Protoc,1999,3(3):242-247.
[89]郝敏,谷守芹,韩建民,董金皋.cDNA末端扩增技术的研究进展.河北林果研究,2006,21(2)：157-161.
[90]Ewards J, Delort J, Mallet J. Oligodeoxyribonucleotide ligation to single-stranded cDNAs:A new tool for clonig 5'ends of mRNA and for contructing cDNA libraries by in vitro amplification[J].Nucleic Acids Res,1991,19(19):5227-5232.
[91]Maruyama N, Rakow T. L, Maruya ma H. IcRACE:A simple method for identification of the 5'ends of mRNAs. Nucleic Acids Res,1995,23(18):3796-3797.
[92]Liu X. W, Gorovsky M. A. Mapping the 5'and3'ends of Tetrahymena thermophila mRNA using RNA ligase mediated amplification of cDNA ends(RLM-RACE). Nucleic Acids Res,1993, 21(21):4954-4960.
[93]Liao J. H, Hung C. C, Lee J. S, et al. Characterization、Cloning and Expression of porcine ab Crystallin. Biochem Biophys Res Commun,1998,244(1):131-137.
[94]赵乃刚,堵南山,包样生等.河蟹的人工繁殖与增养殖.合肥：安徽科学技术出版社.1988：1-234.
[95]中国渔业统计年监(2000年).农业部渔业局.北京：农业出版社,2001.1-141.
[96]贺竹梅,刘秋云.一种提取质粒DNA的改良法.生物技术,1996,6(1)：37-38.
[97]姜宏,李麓芸.PCR法直接筛选重组阳性克隆.安徽医学,2005,26(2)：89-90.
[2]金刚.早熟河蟹的养殖生态学及渔业价值评估.水生生物学报,1998,22(2)：143-147.
[3]杨洪良.稻田养扣蟹性早熟原因及对策.中国水产,1994,(11)：24-25.
[4]柳志会,孙福新.中华绒螯蟹池塘养殖性早熟产生原因的初步分析与其预防.海水养殖1992,(1-2)：66-69.
[5]陈贤龙,沈江平.一龄河蟹性早熟现象观察.水产科技情报,1999,26(1)28-31.
[6]何正侃,印骏,朱雅珠.密度、营养与河蟹蟹种生长及性早熟之间的关系.水产科技情报,1999,26(2)：73-76.
[7]徐如卫,江锦坡,陆开宏等.河蟹性早熟原因的初步研究.浙江海洋学院学报(自然科学版),2001,20(3)：196-198.
[8]丛宁,臧素娟,凌爱珍等.河蟹研究的若干进展.内陆水产,1994,11：2-5.
[9]王勇军.周期性温度变化对幼蟹生长发育的影响.水产养殖,1999,4：11-20.
[10]赵晓红,送笛.温度、降盐度及pH对中华绒螯蟹幼体变态的影响.大连水产学院学报2001,16(4)：249-256.
[11]吴琅虎,李杰,温周瑞等.河蟹性早熟产生原因及预防措施的探讨.内陆水产,1999,6：15-18.
[12]胡兆群.控制河蟹性早熟技术研究.河北渔业,1998,5：9-11.
[13]朱雅珠,张根玉,王建军等.饲料中适宜的动植物蛋白比与河蟹蟹种生长及性早熟的相关.水产科技情报,1999,26(1)：59-63.
[14]李云峰,康现江,赵晓瑜等.河蟹性早熟发生的相关内在因素研究进展.水产科学,2005,24(2)：34-36.
[15]周开亚,高志千.RAPD标记鉴别中华绒螯蟹种群初步研究.应用与环境生物学报,1999,5(2)：176-180.
[16]张德隆,杜晓燕,赵金利等.河蟹性早熟成因及控制方法研究.淡水渔业,2001,31(4)：36-39.
[17]羊茜,占家智.辽河蟹与长江蟹生长速度的对比试验.淡水渔业,1999,29(10)：40-42.
[18]孙金生,刘安西,杜育哲等.中华绒螯蟹窦腺的显微和超微结构.动物学报,2001,47(1)：27-31.
[19]赵维信,陆剑峰.中华绒螯蟹大颚器官激素生物合成与性早熟的关系.水产学报,2003,27(4)： 289-294.
[20]赵维信,陆剑峰.中华绒螯蟹Y-器官在蜕皮周期中的超微结构.中国水产科学,2004,11(1)：74-76.
[21]吴嘉敏,姜新耀.中华绒螯蟹血淋巴钙离子和17p-雌二醇浓度与性早熟的关.水产学报,2001,25(2)：112-115.
[22]成永,旭王武.虾蟹类脂类营养与中华绒鳌蟹性早熟.科学养鱼,2000,6：39-40.
[23]吴嘉敏,姜新耀.中华绒螯蟹血淋巴和肝胰腺的总蛋白含量与性早熟的关系.水产学报,2000,24(4)：306-311.
[24]陈再忠,成勇旭,王武.早熟期间中华绒螯蟹肝胰腺指数、肝脂含量及脂肪酸组成的变.水产学报,2003,27(1)：57-61.
[25]康现江,米娅,孙辉建等.中华绒螯蟹眼柄神经内分泌系统结构的研究.华东师范大学学报(自然科学版),1998：22-26.
[26]康现江,郭明申,米娅等.中华绒螯蟹眼柄神经分泌系统结构的研究：Ⅲ.窦腺的显微和超微结构.中国动物科学研究,1994：103-106.
[27]赵维信,贾江,安苗.外源激素和眼柄提取物对罗氏沼虾卵目细胞的离体诱导作用.上海水产大学学报,1996,5(4)：221-225.
[28]堵南山,赖伟,安婴等.中华绒螯蟹受精的细胞学研究.中国科学(B辑),1992,36(3)：260-265.
[29]王在照,相建海.甲壳动物CHH家族神经激素结构和功能研究进展.水产学报,2001,25(2)：175-180.
[30]Kleijn P.V. De and Franqois V. H. Molecular biology of neurohormone precursors in the eyestalk of Crustacea. Comp. Biochem. Physiol,1995,112 (4):573-579.
[31]Gu P.L, Chan S. M. Cloning for a cDNA encoding a putative Molt-Inhibiting Hormone from the eyestalk of the sand shrimp Metapenaeus ensis. Molecular Marine Biology and Biotechnology,1998, 7(3):214-220.
[32]Soyez D, LeCaer J. P, Noel P Primary structure of two isoforms of the vitellogenesis-inhibiting hormone from the lobster Homarus americanus. Neuropeptides 1991.20,25-32.
[33]Aguilar, M. B, Quackenbush, L. S, Hunt, D. T, et al. Identification, purification and initial characterization of the vitellogenesis-inhibiting hormone from the Mexican crayfish Procambarus bouOieri Ortmann. Comp. Biochem. Physiol.1992.102B,491-498.
[34]Keller R. Crastacean neuropeptides:structures, functions and comparative aspects, experimenti-al,1992,48(5):439-448.
[35]蔡生力.甲壳动物内分泌学研究与展望.水产学报,1998,22(2)：154-161.
[36]Kegel G, Reicwein B, Weese S, et al. Amino acid sequence of Crustacean Hyperglycemic Hormore(CHH) from the shore crab Carcinus maenas. FEBS Lett,1989,255:10-14.
[37]Tensen C. P, Keijn D. P., Van H. F. Cloning and sequence analysis of cDNAs encoding two crustacean hyperglycemic hormore in the lobster, Homarus americanus. Eur J Biochem,1991,200:103-106.
[38]Martin G, Sorokine O, Van D. A. Isolation and molecular characterization of a hyperglycemic neuropetide from the sinus gland of the terrestial isopod, Armadillidium vulgare (Crustacea). Eur J Biochem,1993,211:601-607.
[39]Kegel G, Reicwein B, Tense C. P, et al. Amino acid sequence of Crustacean Hyperglycemic Hormore(CHH) from the crayfish Orconectes limosus. Emergence of a novel neuropeptide family. Peptides,1991,12:909-913.
[40]Huberman A, Aguilar M B, Brew K, et al. Primary structure of the major isomorph of Crustacean Hyperglycemic Hormore(CHH-1) from the sinus gland of the Mexican crayfsh Procambarus bouvieri (Ortmmann):interspecies comparison. Peptides,1993,14:7-16.
[41]Yasuda A, Yasuda Y, Fujita T, et al. Characterization of crustacean hyperglycemic hormore from the crayfish (Procambarus clarkii):Multiplicity of molecular stereoversion and diverse functions. Gen Comp Endocr,1994,95:387-398.
[42]Ohira T, Watanabe T, NagasawaH, et al. Molecular cloning of cDNAs encoding four crustacean hyperglycemic hormones and a molt-inhibiting hormone from the kuruma prawn Penaeus japonicus. Proceedings of the ⅩⅢ international congress of comparative endocrinology. Yokohama,1998,83-86.
[43]Chung J. S, Wilkinson M C, Webster S G Amino acid sequences of both isoforms of crustacean hyperglycemic hormone(CHH) and corresponding precursor-related peptide in Cancer Pagurus. Regul Pept,1998,7:17-24.
[44]Gu P. L, Chan S M. The shrimp hyperglycemic hormone-like neuropeptide is encoded by multiple copies of genes arranged in a cluster. FEBS Lett,1998,441:397-403.
[45]Marco H. G, Brandt W, Gaede G. Elucidation of the Amino Acid Sequence of a Crustacean Hyperglycemic Hormone from the Spiny Lobster, Jasus lalandii Bioch BioPhys Res Comrnun, 1998,248:578-583.
[46]Sithigorngul W., Jaideechoey S., Saraithongkum W, et al. Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.J Exp Zool,1999,284:217-224.
[47]Ju B, Khoo H. W. Characterization of crustacean hyperglycemic hormone(CHH) mRNA transcripts and genomic sequences in Macrobrachium lanchesteri.1998. Submitted to the EMBL/GenBank/DDBJdatabase.
[48]Davey M. L, Hall M. R, Willis R H,et al.Five crustacean hyperglycemic family hormones of Penaeus monodon:complementary DNA sequence and identification in single sinus glands by electrospray ionization-fourier transform mass spectrometry.Mar Biotechnol,2000,2:80-91.
[49]Huberman. Secordary structure of a crustacean neuropeptide hormone family by means of CD. Peptides,1989,10:1113-1115.
[50]Mattson M. P. E. spaziani.5-Hydroxy-trytamine mediates release of molt-inhibiting hormone activity from isolated crab eyestalk ganglia.Bio Bull,1985,169:246-255.
[51]Ohira T, Watansbe T. Molecular cloning of a molt-inLhibiting hormone form the kuruma prawn Penaeus japonicus. Zoological Science,1997,14:785-789.
[52]Mark P, Mattson, Eugene, et al. Regulation of crab y-organ steroidogenesis invitro:evidence that ecdysteroid production increases through activation of cAMP-phosphdiesterase by calcium-calmodalin. Molecular and cellular Endocrinology,1986,48:135-151.
[53]Lachaise F., Roux A. Le, Hubert M. The molting gland of crustaceans:localization, activity and endocrine control. Crustacean Bio,1993,13(2):198-234.
[54]Sedlmeier. Regulation of ecdysteroid biosynihesis in crayfish y-organ.1 Role of cyclic nucleotides. Exp.2001,1993,265:448-453.
[55]Nagasawa H.Isolation and amino acid sequence of a molt-inhibiting hormone from the American crayfish, Procambarus clartii. Biosci Biotech.Biochem,1996,60:554-556.
[56]Klein J. M., Mangerich S., Kleijn D. P. V, et al. Molecular cloning of crustacean putitive molting-inhibiting hormone(MIH)Precursor. FEBS,1993,334:139-142.
[57]Sun P. S. Molecular cloning and sequence analysis of a cDNA encoding a molt-inhibiting hormone-like neuropeptide from the white shrimp Penaeus vannamei. Mol Mar Biol Biotech,1994, 3:1-6.
[58]Lee K. J. Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus.Biochem Biophys Res Commun,1995,209:1126-1131.
[59]Chung J. S., Wilkinson M. C., Webster S. G. Determination of the amino acid sequence of the molt-inhibiting hormone from the edible crab, Cancer pagurus.Neuropeptides,1996,30:95-101.
[60]Aguilar M. B., Falchetto R, ShabanowitzJ, et al. Complete primary structure of the molt-inhibiting honnone of the Mexican crafish Procambarus bouvieri. Peptide,1996,17(3):367-374.
[61]Umphrey H. R., Lee K. J., Watson R. D., et al. Molecular cloning of a cDNA encoding molt-inhibiting hormone of the crab, Cancer magister.Mol Cell Endocrinol,1998,136:145-149.
[62]王在照,相建海,崔朝霞.编码中华绒螯蟹蜕皮抑制激素基因的cDNA片段克隆和序列分析.海洋与湖沼,2002,33(4)：432-438.
[63]王在照,焦传珍,张晓军等.中国对虾蜕皮抑制激素全长cDNA的克隆及序列分析.遗传学报,2003,30(2)：128-134.
[64]王在照,相建海.编码鹰爪虾蜕皮抑制激素基因的cDNA片段的克隆和序列分析.水产学报,2002,26(6)：487-492.
[65]Meusy J. J., Martin G., Soyez D., et al. Immunochemical and immunocytochemical studies of the crustacean vitellogenesis-inhibiting hormone (VIH). Gen Comp Endocrinol,1987,67(3):333-41.
[66]Kleijn D.P., Janssen K.P.,Waddy S.L, et al. Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American Lobster Homarus americanus J Endocrinol.1998,156(2):291-298.
[67]Edomi P., Azzoni E., Mettulio R., et al. Gonad-inhibiting hormone of the Norway Lobster(Nephrops norvegicus):cDNA cloning,expression,recombinant protein production,and immunolocalization.Gene, 2002,284,93-102.
[68]顾志敏,何林岗.中华绒螯蟹发育周期的组织学细胞学观察.海洋与湖沼,1997,28(2)：138-145.
[69]陈炳良,堵南山.中华绒螯蟹幼体中肠的超微结构.海洋与湖沼,1992,23(5)：533-535.
[70]堵南山,陈炳良,赖伟.中华绒螯蟹幼体消化系统发育的研究.海洋与湖沼,1992,23(1)：79-82.
[71]顾志敏,何林岗.切除单侧眼柄对中华绒螯蟹蜕皮、生长、成熟的影响.淡水渔业,1991,5：10-13.
[72]康现江.中华绒螯蟹眼柄神经内分泌系统结构的研究：Ⅲ窦腺的显微和超微结构,中国动物科学研究,1994：103-106.
[73]康现江,米娅,刘彬彬等.摘除眼柄对中华绒螯蟹精巢发育及其氨基酸含量的影响.台湾海峡,2000,19(3)：360-364.
[74]赵维信.十足目甲壳动物生殖激素对卵巢得作用及其调控.上海水产大学学报,200110(2)：166-171.
[75]林远声,邓锋,廖家遗.罗氏沼虾眼柄性腺抑制激素(GIH)的分离纯化及性质的研究.中山大学学报(自然科学版),2000,39(2)：93-95.
[76]Adiyodi, R. G, Bliss D. E, Mantel, et al. Reproduction and its control. In The Biology of Crustacea Academic Press, New York,1985,9:147-215.
[77]Weerawan S., Sompong J, Wanpen S., et al. Purification and characterization of an isoform of Crustacean Hyperglycemic Hormone from the eyestalk of Macrobrachium rosenbergii. Journal of Experimental Zoology,1999,284:217-224.
[78]Soyez D, Deijinen J. E, Martin M. Isolation and characterization of a vitellogene-sis-inhibiting factor from sinus glands of the lobster Homarus americanus.J Exp 2001,1987,244:479-484.
[79]Soyez D, Lecaer J. P, Noel P. Y, et al. Primary structure of two isoforms of the vitellogenesis-inhibiting hormone from the lobster Homarus americanus. Neuropeptides 1991.20,25-32.
[80]Fingerman M. Inhibition of ovarian growth by cadmium in the fiddler crab, Ucapugilator (Decapoda, ocypodidae). Ecotoxicol Environ Saf.,2000,46(2):202-206.
[81]Kleijn D. P., Sleutels F. J, Martens G. J, et al. Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone(GIH) in the lobster Homarus americanus.FEBS Lett,1994, 353:255-258.
[82]Edomi P., Azzoni E., Mettulio R., et al. Gonad-inhibiting hormone of the Norway Lobster(Nephrops norvegicus):cDNA cloning,expression, recombinant protein production,and immunolocalization.Gene 2002,284,93-102.
[83]Gu S, Tobe B. K, et al. Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis Peptides,2002,23:1875-1883.
[84]Ollivaux, C., Vinh J., Soyez D, et al. Crustacean hyperglycemic hormone family peptides in the lobster Homarus gammarus:occurence of genetic variants and posttranslationally modified isoforms GenBank DQ181793.2005.
[85]张庆华,茅矛,陈竺.基因组研究中全长cDNA克隆的策略.生物工程进展,2000,20(4)：3-5.
[86]Lohey J, Ellotf J, Cwirlas J, et al. Polymerase chain reaction with single-sided specificity:analysis of T cell receptor delta chain. Science,1989,243(488):217-222.
[87]Oharao J. H, Doritrl J, Gilbert C. Direct genomic sequencing of bacterial DNA:the pyruvate kinase 1 gene of Escherichia coli. Proc Natl Acad Sci USA,1989,86(18):6883-6887.
[88]Fehrc C, Fickova M, Hiemke C, et al. Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase6:an improved method to determine 5'-and3'-cDNA ends. Brain Res Protoc,1999,3(3):242-247.
[89]郝敏,谷守芹,韩建民,董金皋.cDNA末端扩增技术的研究进展.河北林果研究,2006,21(2)：157-161.
[90]Ewards J, Delort J, Mallet J. Oligodeoxyribonucleotide ligation to single-stranded cDNAs:A new tool for clonig 5'ends of mRNA and for contructing cDNA libraries by in vitro amplification[J].Nucleic Acids Res,1991,19(19):5227-5232.
[91]Maruyama N, Rakow T. L, Maruya ma H. IcRACE:A simple method for identification of the 5'ends of mRNAs. Nucleic Acids Res,1995,23(18):3796-3797.
[92]Liu X. W, Gorovsky M. A. Mapping the 5'and3'ends of Tetrahymena thermophila mRNA using RNA ligase mediated amplification of cDNA ends(RLM-RACE). Nucleic Acids Res,1993, 21(21):4954-4960.
[93]Liao J. H, Hung C. C, Lee J. S, et al. Characterization、Cloning and Expression of porcine ab Crystallin. Biochem Biophys Res Commun,1998,244(1):131-137.
[94]赵乃刚,堵南山,包样生等.河蟹的人工繁殖与增养殖.合肥：安徽科学技术出版社.1988：1-234.
[95]中国渔业统计年监(2000年).农业部渔业局.北京：农业出版社,2001.1-141.
[96]贺竹梅,刘秋云.一种提取质粒DNA的改良法.生物技术,1996,6(1)：37-38.
[97]姜宏,李麓芸.PCR法直接筛选重组阳性克隆.安徽医学,2005,26(2)：89-90.