凡纳滨对虾ANT2 基因的克隆及低温表达谱分析
MOLECULAR CLONING OF LVANT2 GENE AND ITS EXPRESSION PATTERN BY COLD INDUCTION
-
摘要: 为研究凡纳滨对虾适应低温的分子机理, 实验对从低温处理凡纳滨对虾抑制性消减文库中筛选出的一个360 bp EST 序列进行了研究。首先, 同源比对显示该EST 片段与其他物种的ANT2 基因高度同源, 命名为凡纳滨对虾ANT2 基因(LVANT2); 其次, 通过构建凡纳滨对虾肝胰腺全长cDNA 文库, PCR 扩增获得LVANT2 基因的全长cDNA1540 bp, 其中包括1011 bp 的完整开放阅读框, 编码336 个氨基酸残基。然后, 对基因进行了不同组织和低温处理的表达谱分析: (1)组织表达谱的结果显示, 该基因在凡纳滨对虾肌肉组织中表达量最高; (2)在不同低温处理下的表达结果显示, 该基因在15℃处理下基因表达量发生显著变化, 13℃开始呈下调表达, 11℃时表达量又升高; 13℃低温处理不同时间发现该基因在12h 内表达量发生显著变化,48h 后表达量最高。LVANT2 基因的低温诱导表达模式说明其可能在凡纳滨对虾低温适应中发挥作用Abstract: To study the molecular mechanism of cold tolerance of Litopenaeus`vannamei, a 360 bp EST was isolated from suppression subtractive hybridization library of hepatopancreas which treated by low temperature. The 360 bp EST shared highly homology with ANT2 gene of other species, so we named it Litopenaeus vannamei ANT2 gene (LVANT2). Then we cloned whole cDNA sequence of PVANT2 from a hepatopancreas cDNA library. The LVANT2 gene was 1540 bp with a 1011 bp open reading frame encoding a peptide of 309aa. Tissue expression pattern by real-time PCR revealed that LVANT2 mRNA existed in heart, hepatopancreas, muscle, stomach, intestinal and eyestalk but mainly existed in muscle tissue. Cold induction by different temperatures revealed that LVANT2 mRNA expression started to increase after 15℃ treatment for 36 hours, and then decreased after 13℃ treatment for 36 hours, but increased again after 11℃ treatment. As for 13℃ treatment for different times, the expression pattern changed during the first 12 hours, but the expression with low temperature treatment for 24 hours and 36 hours was almost the same with the room temperature. The low temperature induction expression pattern demonstrated that LVANT2 may function in cold tolerance of Litopenaeus vannamei.
-
Keywords:
- LVANT2 /
- Expression /
- SSH library /
- Full-length cDNA library
-
-
[1] Liang P, Pardee A B. Differential display of eukaryotic messengenRNA by means of the polymerase chain reaction ［J］.Science, 1992, 257(5072): 967-971
[2] Hubank M, Schatz D G.. Identifying differences in mRNAexpressing by re-presentational difference analysis of cDNA［J］. Nucleic Acids Research, 1994, 22: 5640-5648
[3] Velculescu V E, Zhang L, Vogelstein B. Serial analysis ofgene expression［J］. Science, 1995, 270(5235): 484-487
[4] Lee S, Tomasetto C, Sager R. Positive selection of candidatetumor-suppressor genes by subtractive hybridization ［J］.Proceedings of the National Academy of Sciences of theUnited State of America, 1991, 88: 2825-2829
[5] Diatchenko L, Lau Y F, Campbell A P, et al. Suppressionsubtractive hybridization: A method for generating differentiallyregulated or tissue-specific cDNA probes and libraries［J］. Proceedings of the National Academy of Sciences of theUnited State of America, 1996, 93(12): 6025-6030
[6] Cai X H, Lu Y S, Wu Z H, et al. Construction of substractedcdna library of litopenaeus vannamei induced by vibrioalginolyticus and analysis of expressed sequence tags ［J］.Acta Hydrobiologica Sinica, 2010, 34(1): 157-163 [蔡小辉,鲁义善, 吴灶和, 等. 溶藻弧菌诱导凡纳滨对虾消减文库的构建及其表达序列标签分析. 水生生物学报, 2010,34(1): 157-163]
[7] Bachem C W, Van Der Hoeven R S, De Bruijin S M, et al.Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP, analysis ofgene expression during potato tuber development ［J］. PlantJournal, 1996, 9: 745-753
[8] Santamaria M, Lanave C, Saccone C. The evolution of theadenine nucleotide translocase family ［J］. Gene, 2004, 333:51-59
[9] Bof M, Brandolin G, Satre M, et al. The mitochondrial adeninenucleotide translocator from Dictyostelium discoideum,functional characterization and DNA sequencing ［J］. EuropeanJournal of Biochemistry, 1999, 259: 795-800
[10] Brand M D, Pakay J L, Ocloo A, et al. The basal protonconductance of mitochondria depends on adenine nucleotidetranslocase content ［J］. Biochemical Journal, 2005, 392:353-362
[11] Lunardi J, Hurko O, Engel W K. The multiple ADP/ ATPtranslocase genes are differentially expressed during humanmuscle development ［J］. The Journal of Biological Chemistry,1992, 267: 15267-15270
[12] Thuswaldner S, Lagerstedt J O, Rojas-Stutz M, et al. Identification,expression, and functional analyses of a thylakoidATP/ADP carrier from Arabidopsis ［J］. Journal of Biochemistry,2007, 282: 8848-8859
[13] Doerner A, Pauschinger M, Badorff A, et al. Tissue-specifictranscription pattern of the adenine nucleotide translocasesisoforms in humans ［J］. FEBS Letters, 1997, 414: 258-262
[14] Kenneth J L, Thomas D S. Analysis of relative gene expressiondata using realtime quantitative PCR and the 2?ΔΔCTmethod ［J］. Methods, 2001, 25: 402-408
[15] Ji W, Wright M B, Cai L, et al. Efficacy of SSH PCR inisolating differentially expressed genes ［J］. BMC Genomics,2002, 20(3): 12
[16] Mao X G, Jing R L, Kong X Y, et al. Comparison of methodsto construct a ful1 length cDNA library ［J］. Hereditas,2006, 28(7): 865-873 [毛新国, 景蕊莲, 孔秀英, 等.几种全长cDNA 文库构建方法比较. 遗传, 2006, 28(7):865-873]
-
期刊类型引用(5)
1. 杨钧渊，陈锦辉，钟俊生，吴建辉，罗渊政，杜志鹏，陈再忠. 长江口崇明东滩水域仔稚鱼种类组成及多样性. 上海海洋大学学报. 2023(04): 829-840 . 
百度学术
2. MA Zuhang，DONG Tianyao，LIAO Wanying，FAN Xinpeng，XU Yuan，GU Fukang. Ciliates in the Marsh Wetlands of Chongming Island: Taxonomy of Five Species and a Species List. Journal of Ocean University of China. 2019(02): 441-454 . 必应学术
3. 吕亭豫，龚政，张长宽，耿亮，张茜. 粉砂淤泥质潮滩潮沟形态特征及发育演变过程研究现状. 河海大学学报(自然科学版). 2016(02): 178-188 . 百度学术
4. 张亚洲，蒋日进，李凯，范正利. 瓯江口水域春、夏季浮游动物群落结构特征. 浙江海洋学院学报(自然科学版). 2016(03): 201-206 . 百度学术
5. 张星烁，郝志，赵鲁安，陈利红，李枫，孙大明，刘彬. 江苏大丰盐沼鸟类群落空间结构的影响因素研究. 野生动物学报. 2015(02): 171-178 . 百度学术
其他类型引用(6)
计量
- 文章访问数:
- HTML全文浏览量: 0
- PDF下载量:
- 被引次数: 11
下载: