嗜水气单胞菌诱导的池蝶蚌血细胞cDNA文库的构建及亲环蛋白基因的序列分析
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摘要
池蝶蚌(Hyriopsis Schlegeli)原产于日本滋贺县的琵琶湖,属软体动物门、瓣鳃纲、蚌科、帆蚌属,与我国三角帆蚌为同属不同种。池蝶蚌无论形态、生长速度、产量、成活率、育珠能力皆优于其它淡水育珠蚌,更具有经济优势,因而国内已大力发展池蝶蚌作为我国的重要育珠对象之一。本研究构建了经过嗜水气单胞菌诱导的池蝶蚌血细胞cDNA全长文库并对部分序列进行了序列分析,并重点对免疫相关基因亲环蛋白(Cyp A)进行了较详细的序列分析。主要结果如下：1.利用灭活的嗜水气单胞菌(Aeromonas Hydrophila)诱导处于旺盛生长期(四龄)的池蝶蚌14h,采用Trizol试剂提取诱导后的池蝶蚌血细胞的总RNA。根据Creator SMART试剂盒用户手册,逆转录后,用LD-PCR合成双链cDNA,并逐步构建成池蝶蚌血细胞的全长cDNA文库。本文库为首次成功构建的淡水贝类池蝶蚌血细胞全长cDNA文库。原始文库的滴度为4×106cfu/cm3,重组率为90%,扩增后文库的滴度为3.55×109pfu/ml。
2.从文库共随机测序102个样品,所得双向测序结果去除载体,拼接,并多序列比对去除重复序列后,余59条已知功能序列,其余为未知功能序列。序列中最小长度270 bp,最大长度为2153bp,平均大小608.6 bp,表明插入片段大小理想。已知基因根据其功能可分为以下几大类：基础代谢类基因,细胞凋亡,细胞分裂调节、蛋白质的装配、修饰与降解,DNA合成、转录、翻译及重组相关基因,核糖体基因和免疫相关基因。
3.从文库中筛选获得免疫相关基因池蝶蚌亲环蛋白A(HsCyp A)全长基因并进行序列分析,池蝶蚌Cyp A基因全长1229个碱基,其在53-547位存在一个长495 bp的开放阅读框,编码164个氨基酸。其起始密码子是atg,终止密码子是taa。HsCypA序列还包括52bp的5'非编码区(untranslated region,UTR),682bp的3'UTR,和29 bp的poly(A)尾,没有明显的加尾信号。ORF编码区蛋白的分子量为17.26 kDa,理论等电点PI=8.9。对Cyp A氨基酸序列二级结构进行了较详细的分析并进行了三维建模,同时构建了其系统进化树,分析表明亲环蛋白家族是一个在进化上非常保守的蛋白家族。
本研究为课题组基础性工作,其研究成果将为大规模筛选文库、发现新的功能基因提供有效的平台,为免疫相关特定基因的研究提供了较好的依据。
Hyriopsis schlegelii originated in Biwa lake of Japan and was introduced to China in 1997. Compared with other freshwater pearl mussel, the Hyriopsis schlegelii has high rate of growth, better disease-resistance and pearling production. Considering economical interests, it is necessary to select breed with better disease-resistance phenotype.
A full-length haemocytes cDNA library was constructed in order to clone genes involving in Aeromonas Hydrophila defenses from Hyriopsis Schlegeli.Inactivated Aeromonas Hydrophila were used to chalenge Hyriopsis Schlegeli (fourth instar). And then we extracted the total RNAs from haemocytes using TRIzol reagent. The"anchor first-strand cDNA"containing a symmetrical sfiI restriction enzyme sites (A&B) was synthesized by transcription of total RNA with the SMART technique.The SMART switching mechanism at 5'end of the RNA transcript technique was used to construct the cDNA library.
The titer of the unamplified cDNA library was 4×106cfu/cm3 with a recombinantrate of 90% and that of amplified one was 3.55×109cfu/cm3. A total of 102 clones was selected to be sequenced and 59 were functional sequences.The shortest sequence was 270bp and longest one was 2153 bp.The average length is 608.6 bp. The results indicated that a high quality library was meet the needs for the consequence gene clone and expressed sequence tag analysis.
Cyclophylin A(Cyp A,) was isolated from cDNA library by screening.The full length of the Hyriopsis Schlegeli Cyp A (HsCyp A)was 1229 bp, Sequence analysis showed that HsCyp A cDNA contained 52bp 5'untranslated region(UTR),682bp 3' UTR and 29 bp poly(A) tail.Which also includes an open reading frame (ORF) of 495 bp and encoded 164 amino acid residues,which doesn't exist signal peptides.It's molecular weight is 17.26 kDa and pI 8.9.We also analysised it's secondary structure and homology-modeled the HsCyp A. A phylogenetic tree showed that they might have similarities in biological functions evolving from the same ancient gene.
2.从文库共随机测序102个样品,所得双向测序结果去除载体,拼接,并多序列比对去除重复序列后,余59条已知功能序列,其余为未知功能序列。序列中最小长度270 bp,最大长度为2153bp,平均大小608.6 bp,表明插入片段大小理想。已知基因根据其功能可分为以下几大类：基础代谢类基因,细胞凋亡,细胞分裂调节、蛋白质的装配、修饰与降解,DNA合成、转录、翻译及重组相关基因,核糖体基因和免疫相关基因。
3.从文库中筛选获得免疫相关基因池蝶蚌亲环蛋白A(HsCyp A)全长基因并进行序列分析,池蝶蚌Cyp A基因全长1229个碱基,其在53-547位存在一个长495 bp的开放阅读框,编码164个氨基酸。其起始密码子是atg,终止密码子是taa。HsCypA序列还包括52bp的5'非编码区(untranslated region,UTR),682bp的3'UTR,和29 bp的poly(A)尾,没有明显的加尾信号。ORF编码区蛋白的分子量为17.26 kDa,理论等电点PI=8.9。对Cyp A氨基酸序列二级结构进行了较详细的分析并进行了三维建模,同时构建了其系统进化树,分析表明亲环蛋白家族是一个在进化上非常保守的蛋白家族。
本研究为课题组基础性工作,其研究成果将为大规模筛选文库、发现新的功能基因提供有效的平台,为免疫相关特定基因的研究提供了较好的依据。
Hyriopsis schlegelii originated in Biwa lake of Japan and was introduced to China in 1997. Compared with other freshwater pearl mussel, the Hyriopsis schlegelii has high rate of growth, better disease-resistance and pearling production. Considering economical interests, it is necessary to select breed with better disease-resistance phenotype.
A full-length haemocytes cDNA library was constructed in order to clone genes involving in Aeromonas Hydrophila defenses from Hyriopsis Schlegeli.Inactivated Aeromonas Hydrophila were used to chalenge Hyriopsis Schlegeli (fourth instar). And then we extracted the total RNAs from haemocytes using TRIzol reagent. The"anchor first-strand cDNA"containing a symmetrical sfiI restriction enzyme sites (A&B) was synthesized by transcription of total RNA with the SMART technique.The SMART switching mechanism at 5'end of the RNA transcript technique was used to construct the cDNA library.
The titer of the unamplified cDNA library was 4×106cfu/cm3 with a recombinantrate of 90% and that of amplified one was 3.55×109cfu/cm3. A total of 102 clones was selected to be sequenced and 59 were functional sequences.The shortest sequence was 270bp and longest one was 2153 bp.The average length is 608.6 bp. The results indicated that a high quality library was meet the needs for the consequence gene clone and expressed sequence tag analysis.
Cyclophylin A(Cyp A,) was isolated from cDNA library by screening.The full length of the Hyriopsis Schlegeli Cyp A (HsCyp A)was 1229 bp, Sequence analysis showed that HsCyp A cDNA contained 52bp 5'untranslated region(UTR),682bp 3' UTR and 29 bp poly(A) tail.Which also includes an open reading frame (ORF) of 495 bp and encoded 164 amino acid residues,which doesn't exist signal peptides.It's molecular weight is 17.26 kDa and pI 8.9.We also analysised it's secondary structure and homology-modeled the HsCyp A. A phylogenetic tree showed that they might have similarities in biological functions evolving from the same ancient gene.
引文
[1]Kato S, Sekine S, et al. Construction of a human full-length cDNA bank[J]. Gene,1994, 150(2):243—250.
[2]王关林,方宏筠.植物基因工程(第二版)[M].北京：科学技术出版社,2002,43-170
[3]Diatchenko L, Lau YC, Campbeu AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries [J].Proc Natl Acad SCi USA.1996.93(12):6025～6030.
[4]Gubler U, Hoffman B J. A simple and very effcient method for generating cDNA library[J].Gene,1983,25:263-269.
[5]Efstathios S. Gonos, Anastasia Derventzi, Marie Kveiborg, et al. Cloning and Identification of Genes That Associate with Mammalian Replicative Senescence[J] Exp Cell Res,1998, 240(1):66-74.
[6]Hakvoort TB.et al. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments[J]. NUcleic Acids Res.1996.24:3478-3480.
[7]Diatchenko L. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries [J].Proc Natl Acad SCi USA.1996.93(12):6025-6030.
[8]Gurskaya NG, Diatchenko L,Chenchik A, et al. Equalizing cDNA Subtraction Based on Selective Suppression of Polymerase Chain Reaction:Cloning of Jurkat Cell Transcripts Induced by Phytohemaglutinin and Phorbol 12-Myristate 13-Acetate[J].Analytical Biochemistry.1996.240:90～97.
[9]Brian R. Wong,Jaerang R, Joseph A.et al. TRANCE Is a Novel Ligand of the Tumor Necrosis Factor Receptor Family That Activates c-Jun N-terminal Kinase in T Cells[J].Biol Chem, 1997,272(40):25190～25194.
[10]Chu ZL. McKinsey TA, Liu L, et al. Suppression of tumor necrosis factor-induced cell death by inhibition of apoptosisc-IAP-2 is under NF-kappa B control[J]. Proe Natl Acad Sci USA.1997.94:10057～10062
[11]Saiki RK.,Scharf S., et al. Enzymatic Amplification Of Beta-Globin Genomic Sequences And Restriction Site Analysis For Diagnosis Of Sickle-Cell Anemia [J].Science,1985,230: 1350～1354
[12]Belyavsky A, Vinogradova T, R～ewsky K. Nucleic Acids Res,1989.17(8):2919～2932.
[13]Fromont Racine M. Bertrand E. Pictet R. et al. Nucleic Acids Res.1993.21:1683～1684
[14]Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, et al. Gene,1997.200(12):149～156
[15]Frohman MA, Dush MK, Martin GR. Proc Natl Acad Sci USA,1988.85:8998～9002.
[16]Roeder T. Solid-phase cDNA library construction, a versatile approach [J].Nucleic Acids Res.1998.26:3451～3452.
[17]Carninci P, Shibata Y, et al. Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res,2000, 10(10):1617—1630.
[18]毛新国,景蕊莲等.几种全长cDNA文库构建方法比较[J].遗传,2006,28(7)：865-873.
[19]董志敏,张宝石等.全长cDNA文库的构建方法[J].中国农学通报,2006,2(2),51-55.
[20]Seki M, Carninci P, NishiyamaY, Hayashizaki Y and Shinozaki K. High-efficiency cloning Arabidopsis full—length cDNA by biotinylated CAP trapper. The plant Journal,1998,15(5): 707～720.
[21]Carninci P, Waki K. et al. a complex transcriptome:the construction of the mouse full-length cDNA encyclopedia. Genome Res,2003,13(6B):1273-1289.
[22]Stapleton M, Liao G, et al. The Drosophila genecollection:Identification of putative full-length cDNAs for 70% of D melanogaster genes. Genome Res,2002,12(8):1294-1300
[23]Kikuchi S, Satoh K, et al. Collection, mapping, and annotation of over 28 000 cDNA clones from iaponica rice Science,2003,301(5631):376—379.
[24]EderyI, Chu L L, SonenbergN, Pelletier J. An efficient strategy to isolate full-·length cDNAs based on an mRNA cap retention procedure(CAPture). Mof CelfBiof,1995,15(6):3363 —3371.
[25]Suzuki Y, Sugano S. Construction of a full-length enriched and a 5'-end enriched cDNA Library using the oligo-capping method. Methods Mol Bio,2003.221(1):73—91.
[26]Clepet C, Le Clainche I, Caboche M Improved full—length cDNA production based on RNA tagging by T4 DNA ligase. Nucleic Acids Res,2004,32(1):e6.
[27]Zhu Y Y, Machleder E M, Chenchik A, Li R, Siebert P D. Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction. Biotechniclues, 2001,30(4):892-897.
[z28]Carninci P, Kvam C, et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics,1996,37(3):327-336
[29]Efimov V A, Chakhmakhcheva O G, et al. Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach. Nucl Acids Res,2001,29(22):4751-4759.
[30]刘志刚,济坤美,高波,等.蒿属花粉cDNA文库的构建和初步鉴定[J].热带医学杂志,2004,4(4)：361-363.
[31]赵亚华.分子生物学教程[M].北京：科学出版社,2004.
[32]金小宝,王婷婷,朱家勇.家蚕幼虫脂肪体cDNA文库的构建及初步鉴定[J].中国人兽共患杂志,2005,21(1)：52-55.
[33]晏慧君,黄兴奇等。cDNA文库构建策略及其分析研究进展[J].云南农业大学学报,2006,2(21),1-6
[34]何东苟,余新炳,吴忠道.全长cDNA文库的构建和新基因全长cDNA克隆的策略[J].热带医学杂志,2003,3(4)：473-476.
[35]杨成君,王军.cDNA文库的构建策略及其应用[J].生物技术通报,2007,1：5-9.
[36]Rondinelli RH. CLAR1, a Novel Gene That Exhibits Enhanced Expression in Advanced Human Prostate Cancer[J].Clin Cancer Res.1999.5(6):15951602.
[37]Victor E. Velculescu. Lin Zhang, Bert Vogelstein. et al. Serial analysis of gene expression [J].Scielice.1995.270:484～487
[38]Sompayrac L. Jane S, Bum TC, et al. Overcoming limitations of the mRNA differential display technique[J]. Nucleic Acids Research,1995.23(21):4738～4739.
[39]Kim MK, Lee BS, In JG, et al. Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf [J].Plant Cell Rep,2006(25):599～606
[40]Jung JD, Park HW., Hahn Y. et al. Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags [J].Plant Cell Rep,2003,22:22～230.
[41]Wan JS. Stephen JS, Ghislaine MC.et al. Cloning differentially expressed mRNAs [J].Nature Biotechnology,1996,14:1685～1691.
[42]李靖,韩本立,黄桂君等.应用抑制消减杂交技术构建肝癌差异表达基因消减cDNA文库[J].第三军医大学学报,2001,23(6)：698-701.
[43]罗志勇,刘水平,陆秋恒等.人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析[J].生命科学研究,2003,71(4)：324-328.
[44]李明芳,郑学勤.开发SSR引物方法之研究动态[J].遗传,2004,26(5)：769-776.
[45]曾庆国,陈艺燕.微卫星位点筛选方法综述[J].生态科学,2005,24(4)：368-372.
[46]Schena M. Shalon D, Davis RW. et al. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray [J].Science.1995,270:467～470.
[47]Schena M. Genome analysis with gene expression microarrays[J].Bioassays.1996.18:427～ 431.
[48]杨传平,等.基因芯片技术研究柽柳NaHC03胁迫下基因的表达生物工程学报[J],2005,21(2)：220-226
[49]祝骥,马文丽,姚汝华.DNA芯片技术与基因表达研究[J],生命科学研究,2000,4(2)：106-111
[50]周永灿,潘金培。贝类细胞和体液的防御的防御机制研究进展[J].水产学报,1997,21(4)：449-454.
[51]徐毛喜.池蝶蚌的引种及繁育技术[J].南昌大学学报,2000,24(专辑)：21-24.
[52]周春花,徐毛喜,吴小平,欧阳珊.池蝶蚌(Hyriopsis Schlegeli)与三角帆蚌(H. Cumingii)若干生物学性状比较研究[J].江西科学,2003,21(2)：122-124.
[53]徐毛喜等.池蝶蚌Hyriopsis schlegelii)与三角帆蚌(H. cumingii)育珠生产对比研究[J].江西水产科学,2005,(4)：27-29.
[54]王静,洪一江,王军花,徐毛喜.不同年龄池蝶蚌(贝)与三角帆蚌同工酶的比较[J].科学技术与工程,2005,(4)204-209.
[55]郑汉丰,张根芳,李家乐等.三角帆蚌、池蝶蚌及其杂交F1代早期形态差异分析[J].上海水产大学学报,2005,14(3)：225-230.
[56]李晓英,董志国,程汉良等.池蝶蚌与三角帆蚌及其杂种F1的同工酶鉴别[J].上海水产大学学报,2006,15(2)：140-143.
[57]谢楠等.三角帆蚌、池蝶蚌及杂交F1代养殖效果与育珠性能的比较[J].上海水产大学学报,2006,15(3)：264-269.
[58]洪一江,余颖,郭红军等.池蝶蚌(Hyriopsis schlegeli)血淋巴的抗菌力、溶菌酶和酚氧化酶活力[J].水生生物学报.2008,32(1)：66-69
[59]郭磊,盛军庆,洪一江等.池蝶蚌(贝)血细胞显微观察[J].水生生物学报.2008,32(6)：840-844
[60]余颖,洪一江,邱齐骏等.池蝶蚌胚胎发育与繁殖季节性腺的观察[J].动物学杂志.2008,43(3)：102-107
[61]罗洁,盛军庆,邱齐峻等.人工选育池蝶蚌的EST和MDH同工酶分析[J].水生态学杂志.2008,1(2)：48-52
[62]郭红军,罗洁,洪一江等.人工选育池蝶蚌的生长及不同世代遗传分析[J].水生生物学 报.2008,32(2)：220-224
[63]郭红军。池蝶蚌与三角帆蚌抗菌酶活性及免疫相关基因表达特性的研究[D].南昌大学.2008
[64]李云娟。池蝶蚌外套膜的组织结构观察和钙调蛋白cDNA的克隆和表达特性分析[D].南昌大学.2009
[65]王淑红,邹志华.副溶血弧菌感染的九孔鲍血细胞均一化全长cDNA文库的构建[J].台湾海峡27(3)：278-285.
[66]刘晓,赵敏,高其康,等.皱纹盘鲍(Haliotis discus hannailno)胚胎RNA分离和cDNA文库构建[J].海洋与湖沼,2006,37(4)：355-365.
[67]郑明刚,孙修勤,张进兴.皱纹盘鲍肝和肾cDNA文库的构建及免疫相关基因的初步分析[J].高技术通讯,2007,17(3)：319-324.
[68]张晓军,王兵,张绍萍,等.中国对虾6种组织cDNA文库的构建[J].海洋学报,2005,27(5)：92-102.
[69]贾锡伟,王艺磊,张子平,等.利用SMART技术构建锯缘青蟹精巢和卵巢的cDNA文库[J].厦门大学学报(自然科学版),2004,43(4)：547-550.
[70]魏玉西,郭道森,李丽,陈皓文。几种海产双壳贝类血淋巴中抗菌物质的诱导及其活性测定[J].海洋科学,2002,26(8)：5-8
[71]Sughura Y, Carninci P, Iton M, et al. Comparative evaluation of 5-end-sequence quality of clones in CAP trapper and other full length cDNA libraries[J]. Gene,2001,263:93-102
[72]Zhu Y Y, Machleder E M, Chenchik A, et al. Recverse transcriptase template switching:a SMATR approach for full-length cDNA library construction[J]. Biotechniques,2001,30(4): 892～897
[73]Wellenreuther R, Schupp I, Poustka A, et al. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones[J].BMC Genomics,2004,5(36): 1-8
[74]Edery I,Chu L L,Sonenberyg N,Pelletier J. An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture) [J].Moecularl Cell Biology,1995,15: 3363～3371
[75]Chenchik A, Moqadam F. Siebert P. RNA:Isolation, Analysis and Synthesis [M]. New York:Wiley-Liss,1996,273～321
[76]Caminci P, Kvam C, Kitamura A, et al. High efficiency full length cDNA cloning by biotinylated CAP trapper[J].Genomics,1996,37(3):327～336
[77]Wolfgang M, Schmid T, Manfred W, et al. CapSelect:A highly sensitive method for 5-CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs[J]. Nucleic Acida Research.1999,27 (21):e31
[78]Fimove V A,Chakhmaldacheva O G. Archdeacon J,ct al.Detection of the 5'-cap structure of messenger RNAs with the use of the CAP-jumping approach [J]. Nucleic Acids Research.2001,29:4751～4759
[79]CarninciP, K, Shiraki T, et al.Targeting a complextranscriptomc:the construction of the mouse full-length cDNA encyclopedia[J].Genome Research.2003,13:1273～1289
[80]Seki M, Caminci P, Nishiyama Y, et al. High-efficiency cloning of Ara-bidopsis full-length cDNA by biotinylated CAP trapper[J]. The Plant Journal,1998,15(5):707～720
[81]Kikuchi S. Satoh K, Nagata T, et al. Collection, mapping and annotation of over 28,000 cDNA clones from japonica rice[J]. Science,2003,301:376-379
[82]肖调义,葛熹凯等.2008.2008年中国水产学会学术年会论文摘要集[C]
[83]AndreasD.Baxevanis.The Moleeular Biology Database Collection:2002 update.Nueleic Acide Research,2002,30(1):1-12
[84]Hall,T.A.BioEdit:a user-frendly biological squence alignment editor and analysis program for Windows95/98/NT. Nucl acids Symp.Ser.,1999,41:95-98
[85]KyteJ.,DoolittleR.F.A sample method for displaying the hydropathic character of aprotein.J.Mol.Biol.1982,157:105-132
[86]Hofmann K., Bucher P., Falquet L.,Bairoch A.The PROSITE database, its status in 1999.Nucleic Acids Res.1997,1343:1-15
[87]Thomas Dandekar, Rainer Konig.Computational methods for the prediction of protein folds.Biochemica et Biophysica Acta,1997,1343:1-15
[88]David T Jones.Progress in protein structure prediction.Current Opinion in Structural Biology, 1997,7:377-387
[89]Jaek Sehonbrun,William J Wedemeyer and David Baker. Protein strueture Predietionin. 2002,12:348-354
[90]Peitsch,M.C. Protein modeling by E-mail.Bio/technolo,1995,13:658-660
[91]Peitseh,M.C.ProMod and swiss-Model:internet-based tools for automatede comparative Protein modelling.Biochem Soc Trans,1996,24:274-279
[92]Guex, N.and peitseh, M.C.SWISS-MODEL and theSwiss-Pdb Viewer:An environment for comparative protein modelling.Electrophoresis,1997,18:2714-2723
[93]N.Saito,and M.Nei.The neignbor joining method:a new method for reconstructing philogenetic trees[J].Mol.Biol.Evol,1987,4:406-425
[94]Helekar SA,Patrick J.Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oli-gomeric receptor expression. Proc[J]. Natl. Acad. Sci. USA,1997,94 (10):5432-5437
[95]Kontopidis,G., Taylor,P. and Walkinshaw,M.D. Enzymatic and structural characterization of non-peptide ligand-cyclophilin complexes. Acta Crystallogr[J]. D Biol. Crystallogr.2004, 60:479-485
[96]AJ Koletsky, MW Harding and RE Handschumacher.Cyclophilin:distribution and variant properties in normal and neoplastic tissues[J].The Journal of Immunology,1986,137 (3):1054-1059
[97]Ryffel B,Woerly G, Greiner B,et al.Distribution of the cyclosporine binding protein cyclop-hilin in humantissues[J]. Immunology,1991,72 (3):399-404
[98]Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace:A web-based environment for protein structure homology modelling[J]. Bioinformatics, 22,195-201.
[99]Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL:an automated protein homology-modeling server[J]. Nucleic Acids Research 31:3381-3385.
[100]Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer:An environment for comparative protein modelling[J]. Electrophoresis 18:2714-2723.
[101]Piotukh K, Gu W, Kofler M, Labudde D, Helms V, Freund C(2005) Cyclophilin A binds to linear peptide motifs containing a consensus that is present in many human proteins[J]. J Biol Chem 280:23668-23674.
[102]Dornan J, Taylor P, Walkinshaw MD (2003) Structures of immunophilins and their ligand complexes[J]. Curr Top Med Chem3:1392-1409.
[103]Mark P, Nilsson LA. molecular dynamics study of cyclophilin A free and in complex with the Ala-Pro dipeptide[J]. Eur Biophys J.2007 (36):213-224.
[104]Eisenmesser EZ, Bosco DA, Akke M, Kern D (2002) Enzyme dynamics during catalysis[J]. Science 295:1520-1523.
[105]Howard BR, Vajdos FF, Li S, Sundquist WI, Hill CP (2003)Structural insights into the catalytic mechanism of cyclophilin A[J].Nat Struct Biol 10:475-481.
[106]Tu HB, Yang WL, Jiang XY, Chen HP, Xiong Q, Wei JW et al (2003) Cloning, sequence analysis and evolutionary conservation of a full-length cDNA encoding cyclophilin A from red stingray Dasyatis akajei[J]. Fish Shellfish Immunol 15:359-366.
[107]Hengming Ke,et al.The Structure and Function of Cyclophilins[J].nature.2000.22(3):134-141
[108]Handschumacher R.E.,Handing M.w.,Rice J.,et al. Cyclophilin-A Specific Cytosolic Binding-Protein For Cyclosporin-A[J].Science,1984;226:544-547
[109]Liu J.,Famer J.D.Jr.,Lane W.S.,et al.Calcineurin Is A Common Target Of Cyclophilin-Cyclosporine-A And FKBP-FK506 Complexes[J].Cell,1991;66:807-815
[110]McCaffrey P.G.,Luo C.,Kerppola T.K.,et al. Isolation Of The Cyclosporine-sensitive T-Cell Transcription Factor NFATP[J]. Science,1993;262:750-754
[111]Baker E.K.,Colley N.J.,Zuker C.S. The Cyclophilin Homolog NINAA Functions As A Chaperone, Forming A Stable Complex In-VIVO With Its Protein Target Rhodopsin [J].EMBO J.,1994;13:4886-4895
[112]Xiaoyan Song, Lingling Wang et al. A cyclophilin A inducible expressed in gonad of zhikong scallop Chlamys farrer[J]ⅰ. Mol Biol Rep.2009.36:1637-1645
[113]Yeh HY, Klesius PH.Channel Catfish, Ictalurus punctatus,cyclophilin A and B cDNA characterization and expressionanalysis. Vet Immunol Immunopathol[J].2008.121:370-377
[114]Qiu L, Jiang S, Huang J, Wang W,et al. Molecular cloning and mRNA expression of cyclophilin A gene in black tiger shrimp (Penaeus monodon) [J]. Fish Shellfish Immunol.2009.26(1):115-121
[2]王关林,方宏筠.植物基因工程(第二版)[M].北京：科学技术出版社,2002,43-170
[3]Diatchenko L, Lau YC, Campbeu AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries [J].Proc Natl Acad SCi USA.1996.93(12):6025～6030.
[4]Gubler U, Hoffman B J. A simple and very effcient method for generating cDNA library[J].Gene,1983,25:263-269.
[5]Efstathios S. Gonos, Anastasia Derventzi, Marie Kveiborg, et al. Cloning and Identification of Genes That Associate with Mammalian Replicative Senescence[J] Exp Cell Res,1998, 240(1):66-74.
[6]Hakvoort TB.et al. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments[J]. NUcleic Acids Res.1996.24:3478-3480.
[7]Diatchenko L. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries [J].Proc Natl Acad SCi USA.1996.93(12):6025-6030.
[8]Gurskaya NG, Diatchenko L,Chenchik A, et al. Equalizing cDNA Subtraction Based on Selective Suppression of Polymerase Chain Reaction:Cloning of Jurkat Cell Transcripts Induced by Phytohemaglutinin and Phorbol 12-Myristate 13-Acetate[J].Analytical Biochemistry.1996.240:90～97.
[9]Brian R. Wong,Jaerang R, Joseph A.et al. TRANCE Is a Novel Ligand of the Tumor Necrosis Factor Receptor Family That Activates c-Jun N-terminal Kinase in T Cells[J].Biol Chem, 1997,272(40):25190～25194.
[10]Chu ZL. McKinsey TA, Liu L, et al. Suppression of tumor necrosis factor-induced cell death by inhibition of apoptosisc-IAP-2 is under NF-kappa B control[J]. Proe Natl Acad Sci USA.1997.94:10057～10062
[11]Saiki RK.,Scharf S., et al. Enzymatic Amplification Of Beta-Globin Genomic Sequences And Restriction Site Analysis For Diagnosis Of Sickle-Cell Anemia [J].Science,1985,230: 1350～1354
[12]Belyavsky A, Vinogradova T, R～ewsky K. Nucleic Acids Res,1989.17(8):2919～2932.
[13]Fromont Racine M. Bertrand E. Pictet R. et al. Nucleic Acids Res.1993.21:1683～1684
[14]Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, et al. Gene,1997.200(12):149～156
[15]Frohman MA, Dush MK, Martin GR. Proc Natl Acad Sci USA,1988.85:8998～9002.
[16]Roeder T. Solid-phase cDNA library construction, a versatile approach [J].Nucleic Acids Res.1998.26:3451～3452.
[17]Carninci P, Shibata Y, et al. Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res,2000, 10(10):1617—1630.
[18]毛新国,景蕊莲等.几种全长cDNA文库构建方法比较[J].遗传,2006,28(7)：865-873.
[19]董志敏,张宝石等.全长cDNA文库的构建方法[J].中国农学通报,2006,2(2),51-55.
[20]Seki M, Carninci P, NishiyamaY, Hayashizaki Y and Shinozaki K. High-efficiency cloning Arabidopsis full—length cDNA by biotinylated CAP trapper. The plant Journal,1998,15(5): 707～720.
[21]Carninci P, Waki K. et al. a complex transcriptome:the construction of the mouse full-length cDNA encyclopedia. Genome Res,2003,13(6B):1273-1289.
[22]Stapleton M, Liao G, et al. The Drosophila genecollection:Identification of putative full-length cDNAs for 70% of D melanogaster genes. Genome Res,2002,12(8):1294-1300
[23]Kikuchi S, Satoh K, et al. Collection, mapping, and annotation of over 28 000 cDNA clones from iaponica rice Science,2003,301(5631):376—379.
[24]EderyI, Chu L L, SonenbergN, Pelletier J. An efficient strategy to isolate full-·length cDNAs based on an mRNA cap retention procedure(CAPture). Mof CelfBiof,1995,15(6):3363 —3371.
[25]Suzuki Y, Sugano S. Construction of a full-length enriched and a 5'-end enriched cDNA Library using the oligo-capping method. Methods Mol Bio,2003.221(1):73—91.
[26]Clepet C, Le Clainche I, Caboche M Improved full—length cDNA production based on RNA tagging by T4 DNA ligase. Nucleic Acids Res,2004,32(1):e6.
[27]Zhu Y Y, Machleder E M, Chenchik A, Li R, Siebert P D. Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction. Biotechniclues, 2001,30(4):892-897.
[z28]Carninci P, Kvam C, et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics,1996,37(3):327-336
[29]Efimov V A, Chakhmakhcheva O G, et al. Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach. Nucl Acids Res,2001,29(22):4751-4759.
[30]刘志刚,济坤美,高波,等.蒿属花粉cDNA文库的构建和初步鉴定[J].热带医学杂志,2004,4(4)：361-363.
[31]赵亚华.分子生物学教程[M].北京：科学出版社,2004.
[32]金小宝,王婷婷,朱家勇.家蚕幼虫脂肪体cDNA文库的构建及初步鉴定[J].中国人兽共患杂志,2005,21(1)：52-55.
[33]晏慧君,黄兴奇等。cDNA文库构建策略及其分析研究进展[J].云南农业大学学报,2006,2(21),1-6
[34]何东苟,余新炳,吴忠道.全长cDNA文库的构建和新基因全长cDNA克隆的策略[J].热带医学杂志,2003,3(4)：473-476.
[35]杨成君,王军.cDNA文库的构建策略及其应用[J].生物技术通报,2007,1：5-9.
[36]Rondinelli RH. CLAR1, a Novel Gene That Exhibits Enhanced Expression in Advanced Human Prostate Cancer[J].Clin Cancer Res.1999.5(6):15951602.
[37]Victor E. Velculescu. Lin Zhang, Bert Vogelstein. et al. Serial analysis of gene expression [J].Scielice.1995.270:484～487
[38]Sompayrac L. Jane S, Bum TC, et al. Overcoming limitations of the mRNA differential display technique[J]. Nucleic Acids Research,1995.23(21):4738～4739.
[39]Kim MK, Lee BS, In JG, et al. Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf [J].Plant Cell Rep,2006(25):599～606
[40]Jung JD, Park HW., Hahn Y. et al. Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags [J].Plant Cell Rep,2003,22:22～230.
[41]Wan JS. Stephen JS, Ghislaine MC.et al. Cloning differentially expressed mRNAs [J].Nature Biotechnology,1996,14:1685～1691.
[42]李靖,韩本立,黄桂君等.应用抑制消减杂交技术构建肝癌差异表达基因消减cDNA文库[J].第三军医大学学报,2001,23(6)：698-701.
[43]罗志勇,刘水平,陆秋恒等.人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析[J].生命科学研究,2003,71(4)：324-328.
[44]李明芳,郑学勤.开发SSR引物方法之研究动态[J].遗传,2004,26(5)：769-776.
[45]曾庆国,陈艺燕.微卫星位点筛选方法综述[J].生态科学,2005,24(4)：368-372.
[46]Schena M. Shalon D, Davis RW. et al. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray [J].Science.1995,270:467～470.
[47]Schena M. Genome analysis with gene expression microarrays[J].Bioassays.1996.18:427～ 431.
[48]杨传平,等.基因芯片技术研究柽柳NaHC03胁迫下基因的表达生物工程学报[J],2005,21(2)：220-226
[49]祝骥,马文丽,姚汝华.DNA芯片技术与基因表达研究[J],生命科学研究,2000,4(2)：106-111
[50]周永灿,潘金培。贝类细胞和体液的防御的防御机制研究进展[J].水产学报,1997,21(4)：449-454.
[51]徐毛喜.池蝶蚌的引种及繁育技术[J].南昌大学学报,2000,24(专辑)：21-24.
[52]周春花,徐毛喜,吴小平,欧阳珊.池蝶蚌(Hyriopsis Schlegeli)与三角帆蚌(H. Cumingii)若干生物学性状比较研究[J].江西科学,2003,21(2)：122-124.
[53]徐毛喜等.池蝶蚌Hyriopsis schlegelii)与三角帆蚌(H. cumingii)育珠生产对比研究[J].江西水产科学,2005,(4)：27-29.
[54]王静,洪一江,王军花,徐毛喜.不同年龄池蝶蚌(贝)与三角帆蚌同工酶的比较[J].科学技术与工程,2005,(4)204-209.
[55]郑汉丰,张根芳,李家乐等.三角帆蚌、池蝶蚌及其杂交F1代早期形态差异分析[J].上海水产大学学报,2005,14(3)：225-230.
[56]李晓英,董志国,程汉良等.池蝶蚌与三角帆蚌及其杂种F1的同工酶鉴别[J].上海水产大学学报,2006,15(2)：140-143.
[57]谢楠等.三角帆蚌、池蝶蚌及杂交F1代养殖效果与育珠性能的比较[J].上海水产大学学报,2006,15(3)：264-269.
[58]洪一江,余颖,郭红军等.池蝶蚌(Hyriopsis schlegeli)血淋巴的抗菌力、溶菌酶和酚氧化酶活力[J].水生生物学报.2008,32(1)：66-69
[59]郭磊,盛军庆,洪一江等.池蝶蚌(贝)血细胞显微观察[J].水生生物学报.2008,32(6)：840-844
[60]余颖,洪一江,邱齐骏等.池蝶蚌胚胎发育与繁殖季节性腺的观察[J].动物学杂志.2008,43(3)：102-107
[61]罗洁,盛军庆,邱齐峻等.人工选育池蝶蚌的EST和MDH同工酶分析[J].水生态学杂志.2008,1(2)：48-52
[62]郭红军,罗洁,洪一江等.人工选育池蝶蚌的生长及不同世代遗传分析[J].水生生物学 报.2008,32(2)：220-224
[63]郭红军。池蝶蚌与三角帆蚌抗菌酶活性及免疫相关基因表达特性的研究[D].南昌大学.2008
[64]李云娟。池蝶蚌外套膜的组织结构观察和钙调蛋白cDNA的克隆和表达特性分析[D].南昌大学.2009
[65]王淑红,邹志华.副溶血弧菌感染的九孔鲍血细胞均一化全长cDNA文库的构建[J].台湾海峡27(3)：278-285.
[66]刘晓,赵敏,高其康,等.皱纹盘鲍(Haliotis discus hannailno)胚胎RNA分离和cDNA文库构建[J].海洋与湖沼,2006,37(4)：355-365.
[67]郑明刚,孙修勤,张进兴.皱纹盘鲍肝和肾cDNA文库的构建及免疫相关基因的初步分析[J].高技术通讯,2007,17(3)：319-324.
[68]张晓军,王兵,张绍萍,等.中国对虾6种组织cDNA文库的构建[J].海洋学报,2005,27(5)：92-102.
[69]贾锡伟,王艺磊,张子平,等.利用SMART技术构建锯缘青蟹精巢和卵巢的cDNA文库[J].厦门大学学报(自然科学版),2004,43(4)：547-550.
[70]魏玉西,郭道森,李丽,陈皓文。几种海产双壳贝类血淋巴中抗菌物质的诱导及其活性测定[J].海洋科学,2002,26(8)：5-8
[71]Sughura Y, Carninci P, Iton M, et al. Comparative evaluation of 5-end-sequence quality of clones in CAP trapper and other full length cDNA libraries[J]. Gene,2001,263:93-102
[72]Zhu Y Y, Machleder E M, Chenchik A, et al. Recverse transcriptase template switching:a SMATR approach for full-length cDNA library construction[J]. Biotechniques,2001,30(4): 892～897
[73]Wellenreuther R, Schupp I, Poustka A, et al. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones[J].BMC Genomics,2004,5(36): 1-8
[74]Edery I,Chu L L,Sonenberyg N,Pelletier J. An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture) [J].Moecularl Cell Biology,1995,15: 3363～3371
[75]Chenchik A, Moqadam F. Siebert P. RNA:Isolation, Analysis and Synthesis [M]. New York:Wiley-Liss,1996,273～321
[76]Caminci P, Kvam C, Kitamura A, et al. High efficiency full length cDNA cloning by biotinylated CAP trapper[J].Genomics,1996,37(3):327～336
[77]Wolfgang M, Schmid T, Manfred W, et al. CapSelect:A highly sensitive method for 5-CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs[J]. Nucleic Acida Research.1999,27 (21):e31
[78]Fimove V A,Chakhmaldacheva O G. Archdeacon J,ct al.Detection of the 5'-cap structure of messenger RNAs with the use of the CAP-jumping approach [J]. Nucleic Acids Research.2001,29:4751～4759
[79]CarninciP, K, Shiraki T, et al.Targeting a complextranscriptomc:the construction of the mouse full-length cDNA encyclopedia[J].Genome Research.2003,13:1273～1289
[80]Seki M, Caminci P, Nishiyama Y, et al. High-efficiency cloning of Ara-bidopsis full-length cDNA by biotinylated CAP trapper[J]. The Plant Journal,1998,15(5):707～720
[81]Kikuchi S. Satoh K, Nagata T, et al. Collection, mapping and annotation of over 28,000 cDNA clones from japonica rice[J]. Science,2003,301:376-379
[82]肖调义,葛熹凯等.2008.2008年中国水产学会学术年会论文摘要集[C]
[83]AndreasD.Baxevanis.The Moleeular Biology Database Collection:2002 update.Nueleic Acide Research,2002,30(1):1-12
[84]Hall,T.A.BioEdit:a user-frendly biological squence alignment editor and analysis program for Windows95/98/NT. Nucl acids Symp.Ser.,1999,41:95-98
[85]KyteJ.,DoolittleR.F.A sample method for displaying the hydropathic character of aprotein.J.Mol.Biol.1982,157:105-132
[86]Hofmann K., Bucher P., Falquet L.,Bairoch A.The PROSITE database, its status in 1999.Nucleic Acids Res.1997,1343:1-15
[87]Thomas Dandekar, Rainer Konig.Computational methods for the prediction of protein folds.Biochemica et Biophysica Acta,1997,1343:1-15
[88]David T Jones.Progress in protein structure prediction.Current Opinion in Structural Biology, 1997,7:377-387
[89]Jaek Sehonbrun,William J Wedemeyer and David Baker. Protein strueture Predietionin. 2002,12:348-354
[90]Peitsch,M.C. Protein modeling by E-mail.Bio/technolo,1995,13:658-660
[91]Peitseh,M.C.ProMod and swiss-Model:internet-based tools for automatede comparative Protein modelling.Biochem Soc Trans,1996,24:274-279
[92]Guex, N.and peitseh, M.C.SWISS-MODEL and theSwiss-Pdb Viewer:An environment for comparative protein modelling.Electrophoresis,1997,18:2714-2723
[93]N.Saito,and M.Nei.The neignbor joining method:a new method for reconstructing philogenetic trees[J].Mol.Biol.Evol,1987,4:406-425
[94]Helekar SA,Patrick J.Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oli-gomeric receptor expression. Proc[J]. Natl. Acad. Sci. USA,1997,94 (10):5432-5437
[95]Kontopidis,G., Taylor,P. and Walkinshaw,M.D. Enzymatic and structural characterization of non-peptide ligand-cyclophilin complexes. Acta Crystallogr[J]. D Biol. Crystallogr.2004, 60:479-485
[96]AJ Koletsky, MW Harding and RE Handschumacher.Cyclophilin:distribution and variant properties in normal and neoplastic tissues[J].The Journal of Immunology,1986,137 (3):1054-1059
[97]Ryffel B,Woerly G, Greiner B,et al.Distribution of the cyclosporine binding protein cyclop-hilin in humantissues[J]. Immunology,1991,72 (3):399-404
[98]Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace:A web-based environment for protein structure homology modelling[J]. Bioinformatics, 22,195-201.
[99]Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL:an automated protein homology-modeling server[J]. Nucleic Acids Research 31:3381-3385.
[100]Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer:An environment for comparative protein modelling[J]. Electrophoresis 18:2714-2723.
[101]Piotukh K, Gu W, Kofler M, Labudde D, Helms V, Freund C(2005) Cyclophilin A binds to linear peptide motifs containing a consensus that is present in many human proteins[J]. J Biol Chem 280:23668-23674.
[102]Dornan J, Taylor P, Walkinshaw MD (2003) Structures of immunophilins and their ligand complexes[J]. Curr Top Med Chem3:1392-1409.
[103]Mark P, Nilsson LA. molecular dynamics study of cyclophilin A free and in complex with the Ala-Pro dipeptide[J]. Eur Biophys J.2007 (36):213-224.
[104]Eisenmesser EZ, Bosco DA, Akke M, Kern D (2002) Enzyme dynamics during catalysis[J]. Science 295:1520-1523.
[105]Howard BR, Vajdos FF, Li S, Sundquist WI, Hill CP (2003)Structural insights into the catalytic mechanism of cyclophilin A[J].Nat Struct Biol 10:475-481.
[106]Tu HB, Yang WL, Jiang XY, Chen HP, Xiong Q, Wei JW et al (2003) Cloning, sequence analysis and evolutionary conservation of a full-length cDNA encoding cyclophilin A from red stingray Dasyatis akajei[J]. Fish Shellfish Immunol 15:359-366.
[107]Hengming Ke,et al.The Structure and Function of Cyclophilins[J].nature.2000.22(3):134-141
[108]Handschumacher R.E.,Handing M.w.,Rice J.,et al. Cyclophilin-A Specific Cytosolic Binding-Protein For Cyclosporin-A[J].Science,1984;226:544-547
[109]Liu J.,Famer J.D.Jr.,Lane W.S.,et al.Calcineurin Is A Common Target Of Cyclophilin-Cyclosporine-A And FKBP-FK506 Complexes[J].Cell,1991;66:807-815
[110]McCaffrey P.G.,Luo C.,Kerppola T.K.,et al. Isolation Of The Cyclosporine-sensitive T-Cell Transcription Factor NFATP[J]. Science,1993;262:750-754
[111]Baker E.K.,Colley N.J.,Zuker C.S. The Cyclophilin Homolog NINAA Functions As A Chaperone, Forming A Stable Complex In-VIVO With Its Protein Target Rhodopsin [J].EMBO J.,1994;13:4886-4895
[112]Xiaoyan Song, Lingling Wang et al. A cyclophilin A inducible expressed in gonad of zhikong scallop Chlamys farrer[J]ⅰ. Mol Biol Rep.2009.36:1637-1645
[113]Yeh HY, Klesius PH.Channel Catfish, Ictalurus punctatus,cyclophilin A and B cDNA characterization and expressionanalysis. Vet Immunol Immunopathol[J].2008.121:370-377
[114]Qiu L, Jiang S, Huang J, Wang W,et al. Molecular cloning and mRNA expression of cyclophilin A gene in black tiger shrimp (Penaeus monodon) [J]. Fish Shellfish Immunol.2009.26(1):115-121