ZHOU Xiu-Xia, HUANG Rong, GUO Qiong-Lin. CONSTRUCTION OF A SUBTRACTIVE cDNA LIBRARY FROM THE INTERNAL ORGANS OF TRIONYX SINENSIS EXPERIMENTALLY INFECTED BY AEROMONAS HYDROPHILA[J]. ACTA HYDROBIOLOGICA SINICA, 2007, 31(4): 509-515.
Citation: ZHOU Xiu-Xia, HUANG Rong, GUO Qiong-Lin. CONSTRUCTION OF A SUBTRACTIVE cDNA LIBRARY FROM THE INTERNAL ORGANS OF TRIONYX SINENSIS EXPERIMENTALLY INFECTED BY AEROMONAS HYDROPHILA[J]. ACTA HYDROBIOLOGICA SINICA, 2007, 31(4): 509-515.

CONSTRUCTION OF A SUBTRACTIVE cDNA LIBRARY FROM THE INTERNAL ORGANS OF TRIONYX SINENSIS EXPERIMENTALLY INFECTED BY AEROMONAS HYDROPHILA

  • Received Date: September 25, 2006
  • Rev Recd Date: March 15, 2007
  • Published Date: July 24, 2007
  • Aeromonas hydrophila is one of the main causative agents resulting in serious infectious diseases of turtles and other animals. To understand anti-infectious response to bacteria in reptile, a subtractlve cDNA library was constructed from the liver, spleen and kidney of Chinese soft-shelled turtle (Trionyx sinensis ) experimentally infected with A. hydrophila T4, using suppression subtractive hybridization (SSH). Experimental turtles were injected intraperitoneally with 1.6 ×10^8 CFU live A. hydrophila T4 and the control turtles were injected with steriled normal saline. The liver, spleen and kidney samples of infectious and control turtles were dissected out immediately, and frozen in liquid nitrogen for isolation of total RNA. About 500 mg tissues containing approximately equal amount of liver, spleen and kidney from infectious and control turtles were used as tester and driver samples, respectively. Total RNA extraction was performed from the two samples, followed by mRNA isolation. Using equal amounts of mRNA (2μg) from tester and driver samples, double-strand cDNA was synthesized and digested with restriction enzyme RsaI for three hours. The RsaI-digested tester was subdivided into two pools, and each was ligated with a different adaptor. The RsaI-digested driver cDNA was not exposed to adaptors. Then an excess of driver cDNA was added to each tester cDNA for the first round of hybridization to enrich for differentially expressed sequences. In the second, the two samples from the first hybridization were mixed together and freshly denatured driver DNA was added to further enrich for differentially expressed sequences. New molecules were formed which consist of differentially expressed cDNAs with different adaptors on each end. Then two rounds of suppression PCR were performed. In the first amplification, only ds cDNAs with different adaptor sequences on each end were selected and exponentially amplified. In the second, nested PCR was used to further reduce background and enrich for differentially expressed sequences. Turtle β-actin gene was used as internal control to estimate the efficiency of subtractive cDNA. In this library, β-actin was subtracted significantly at about 2^10 folds, suggesting that the subtractive cDNA library was successfully constructed. The PCR products were inserted into pMD18-T vector and transformed to competent E. coli DHSa cells to set up a subtracted and normalized PCR fragment library. PCR analysis showed that the inserts were 150-800bp in length. To our knowledge, this is the first report of a subtractive cDNA library constructed from experimentally bacteria infected tissue in reptile. A serials of immune-relevant genes, such as IL-8, CD9, CD59, SAA, and ISG12 were first isolated and cloned in turtles (will report in another paper), and the IL-8, CD9, CD59 and ISG12 genes were also first isolated in reptile. The successfully constructed cDNA library will be essential for rapid isolation of differentially expressed genes related to A. hydrophila infection, and useful for understanding the anti-infectious molecular mechanism in reptile.
  • [1]
    Lu C P. Pathogenic Aeromonas hydrophila and the fish diseases caused by it[J]. Journal of Fisheries of China, 1992, 16 (3):282)288[陆承平. 致病性嗜水气单胞菌及其所致鱼病综述.水产学报, 1992, 16(3): 282)288]
    [2]
    Yu Y R, Chu W H, Li K M. Isolation and identification of the pathogen of red neck disease and red abdominal shell disease ofTrionyx sinesis[J]. Progress in Veteri nary Medicine, 2004,25(2): 102)105[虞蕴 如, 储卫 华, 李克敏. 中华鳖红 脖子、红底板病 的病 原分 离鉴 定. 动 物医 学进 展, 2004,25(2): 102)105]
    [3]
    Yang X L, Zhou J G, Ke F E, et al. Epidemiology intestinal necro-sison haemorrhagic of soft-shelled turtle[J]. Journal of Fisheries ofScience of China, 1998, 5(2): 73)78[杨先乐, 周剑光, 柯福恩, 等. 中华鳖出血性肠道坏死症流行病学. 中国水产科学,1998, 5(2): 73)78]
    [4]
    Sun P F, CaiW Q. Pathology of the caverned disease of Chinese soft-shelled turtle[J]. Journal of Fisheries of China, 1996, 20 (2):120)124[孙佩芳, 蔡完其. 鳖穿孔病的病原研究. 水产学报,1996, 20(2): 120)124]
    [5]
    Zhou J G, Yang X L, Ai X H. Pathogen of the furunculosis, red neck and red spot complicating diseases of Trionyx sinensis[J].Journal of Fisheries of Chi na, 1999, 23(3): 270)277[周剑光,杨先乐, 艾晓辉. 中华鳖疖疮、红脖子、赤斑病并发症的病原研究. 水产学报, 1999, 23(3):270)277]
    [6]
    Lu H D, Jin L H. Studies on the Aeromonas hydrophila septicemia of so-ft shelled turtle (Trionyx Sinensis)[J]. Journal of Fisheries ofChina, 1996, 20(3): 223)234[陆宏达, 金丽华. 鳖嗜水气单胞菌败血症的研究. 水产学报, 1996,20(3): 223)234]
    [7]
    Allan B J, Stevenson R M W. Extracellular virulence factors of Aeromonas hydrophila in fish infections[J]. Can J Microbiol, 1981,27: 1114)1122
    [8]
    Shi L, Li L, Zhu Q H, et al. Determine on susceptibility of pathogenic bacteria in sof-t shelled turtle (Pelodisclls sinensis) to thir-teen antimicrobial drugs[J]. Journal of Henan Normal University (Natural Science), 2002, 30 (2): 99)104[石灵, 李莉, 朱庆红, 等. 中华鳖常见致病菌对 13 种抗菌药物的敏感性测定.河南师范大学学报(自然科学版), 2002, 30(2): 99)104]
    [9]
    Yang X L, Ke F E, Liu Z Q. Susceptibility of childish soft-shelled turtle to medicines commonly used[J]. Journal of Fishery Sciencesof China, 1996, 3(2): 58)64[杨先乐, 柯福恩, 刘仲琪. 中华鳖稚鳖对几种药物敏感性的研究. 中国水产科学, 1996,3(2): 58)64]
    [10]
    Yang X L, Ke F E, Ye C G. The turtle diseases and their prevention and control[M]. Beijing: Agriculture Science Technology Press.1995, 97)101[杨先乐, 柯福恩, 叶重光. 鳖病及其防治. 北京: 农业科学技术出版社. 1995, 97)101]
    [11]
    Nie L W, Shan X N, Wang M, et al. The conservative region se-quence analysis of four Sox genes in the Trionyx sinensis[J]. Acta Hydrobiol ogica Sinica, 2001, 25 (3): 245)250[聂刘旺, 单祥年,汪鸣, 等. 中华鳖 4 个 Sox 基因保守区的序列分析. 水生生物学报,2001, 25(3): 245)250]
    [12]
    Venkatesh B, Erdmann M V, Brenner S. Molecular synapomor-phies resolve evolutionary relationships of extant jawed vertebrates[J]. Proc Natl Acad Sci. USA, 200 1, 98 (2 0): 11 382)11387
    [13]
    Kuraku S, Ishijima J, Nishid-aUmehara C, et al. cDNA-based gene mapping and GC3 profiling in the soft-shelled turtle suggest a chro-mosomal size-dependent GC bias shared by sauropsids[J]. Chromo-some Res, 2006, 14: 187)202
    [14]
    Zheng J F, Zhu M Y. Construction and characterization of a cDNA li-brary from kidney/ urogenital complex tissues of on-eweek-old em-bryos of the soft-shelled turtle Trionyx sinensis[J]. Acta Zoologica Sinica, 2005, 51(6): 1156)1161[郑济芳, 朱睦元. 中华鳖孵化一周胚胎的肾脏/ 尿生殖嵴混合组织的 cDNA 文库构建与特征. 动物学报, 2005, 51(6):1156)1161]
    [15]
    Kuraku S, Usuda R, Kuratani S. Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatorygenes in carapace evolution[J]. Evol. Dev, 2005, 7: 1, 3)17
    [16]
    Diatchenko L, Lau Y-F C, Campbell A P, et al. Suppression sub-tractive hybridization: a method for generating differentially regulatedor tissue-specific cDNA probes and libraries[J]. Proc Natl Acad Sci USA, 1996, 93(12): 6025)6030
    [17]
    Tsoi S C M, Ewart K V, Penny S, et al. Identification of immun-erelevant genes from Atlantic salmon using suppression subtractive hy-bridization[J]. Mar. Biotechnol., 2004, 6(3): 199)214
    [18]
    Abdalla S A, Horiuchi H, Furusawa S, et al. Molecular study on chicken tumor necrosis factor recepto-rII and tumor necrosis factor re-cepto-r associated facto-r5[J]. Vet Immunol Immunop, 2004,98(1)2): 31)41
    [19]
    Jolita J U, Thomas J S, Zhao S H, et al. Analysis of porcine diffe-rential gene expression following challenge with Salmonella entericaserovar Choleraesuis using suppression subtractive hybridization[J].Vet Microbiol, 2006, 114(1)2): 60)71
    [20]
    Zhang Y B, Shi Y H, Gui J F. Construction of antiviral subtractive cDNA library of cultured fish cells[J]. Acta Hydrobiologica Sinica,2003, 27(2): 113)118[张义兵, 石耀华, 桂建芳. 鱼类培养细胞抗病毒基因差减 cDNA 文库的构建. 水生生物学报,2003, 27(2): 113)118]
    [21]
    Lu Q, Ren R W, Wang W D, et al. Detection of the aerolysingene in Aeromonas hydrophila by the polymerase chain reaction[J]. Chinese Journal of Veterinary Science, 2 001, 21 (4):347)34 9[卢强, 任瑞文, 王文东, 等. 嗜水气单胞 菌气溶素基因 PCR 检测方法的建立. 中国兽 医学报, 2001,21(4): 347)349]
    [22]
    Cascon A, Fregeneda J, Aller M, et al. Cloning, characterization,and insertional inactivation of a major extracellular serine proteasegene with elastolytic activity from Aeromonas hydrophila[J]. J Fish Dis, 2000, 23: 49)59
    [23]
    Chu W H, Lu C P. Cloning and sequence analysis of an extracellular serine-protease gene of Aeromonas hydrophila J-1[J]. Journal of Fisheries of China, 2004, 24(1): 84)88[储卫华, 陆承平. 嗜水气单胞菌 -J1 株丝氨酸蛋白酶基因克隆与序列分析. 水产学报, 2004, 24(1): 84)88]
    [24]
    Pollard D R, Johnson W M, Lior H, et al. Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction[J].J Clin Microbi ol, 1990, 28(11): 2477)2481
    [25]
    Litman G W, Murphy K, Berger L, et al. Complete nucleotide se-quences of three VH genes in Caiman, a phylogenetically ancientreptile: evolutionary diversification in coding segments and variation in the structure and organization of recombination elements[J].Proc. Natl. Acad. Sci. USA, 1985, 82(3): 844)848
    [26]
    Fritzinger D C, Petrella E C, Connelly M B, et al. Primary structure of cobra complement component C3[J]. J. Immunol., 1992,149(11): 3554)3562
    [27]
    Turchin A, Hsu E. The generation of antibody diversity in the turtle[J]. J. Immunol., 1996, 156(10): 3797)3805
    [28]
    Xia. C Molecular cloning and sequencing analysis of MHC class soft-shelled turtle (Trinyx sinensis)[J]. Chinese Journal of Im-munology, 199 9, 15(2): 77)79[夏春. 中华鳖 MHC I a2链基因克隆及序列分析. 中国免疫学杂志, 1999,15(2): 77)79]
    [29]
    Miller H C, Belov K, Daugherty C H. Characterization of MHC classògenes from an ancient reptile lineage, Sphenodon(tuatara)[J].Immunogenetics, 2005, 57(11): 883)891
    [30]
    Miller H C, Belov K, Daugherty C H. MHC Class I Genes in the tu-atara (Sphenodon spp.): evolution of the MHC in an ancient repti-lian order[J]. M ol. Biol. Ev ol, 2006, 23(5): 949)956
  • Cited by

    Periodical cited type(2)

    1. 朱雨新,李云梅,张玉,王怀警,蔡小兰,成鑫,吕恒. 基于遥感反射率的太湖优势藻识别方法. 湖泊科学. 2023(01): 73-87 .
    2. 褚乔,张壹萱,张玉超,马荣华,胡旻琪. 基于水华蓝藻固有光学特性的主要类群定量识别方法. 湖泊科学. 2021(01): 74-85 .

    Other cited types(5)

Catalog

    Article views PDF downloads Cited by(7)
    Related

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return