烟草叶片全长cDNA文库构建及EST序列分析

doi:10.3864/j.issn.0578-1752.2012.09.004

摘要/Abstract

摘要： 【目的】构建烟草叶片全长cDNA文库并测序，发掘与烟叶发育、代谢和抗逆相关的基因，为进一步研究功能基因奠定基础。【方法】以烟草苗期叶片为试验材料，利用改进的Cap-trapper法构建烟草叶片全长cDNA文库。利用测序技术获得大量的EST序列，采用生物信息分析手段，对所测EST进行拼接和功能注释。【结果】构建了烟草叶片的全长cDNA文库，该文库滴度为1.2×106 pfu•mL-1，平均插入片段在1.4 kb左右。利用该文库测序了5 280个克隆，获得5 233条高质量表达序列标签（EST），序列拼接出3 922个单一基因；同源比对分析表明，89.7%的基因与已知功能基因具有较高的同源性，这些基因涉及细胞生长、信号转导、翻译合成、抗逆反应和能量代谢等功能；并详细鉴定了部分与烟碱合成和抗病相关的基因。【结论】通过Cap-trapper法成功构建了高质量的烟草幼苗叶片全长cDNA文库；大规模EST测序分析挖掘到大量与烟草幼苗叶片发育、抗逆、抗病、烟碱代谢相关的侯选基因。

关键词: 烟草, 全长cDNA文库, 表达序列标签, 功能注释

Abstract: 【Objective】 The full-length cDNA library from tobacco leaves mRNA was constructed. EST data were generated and these gene families of development, metabolizability and resistance were detected and ananlyzed. 【Method】 The Cap-trapper method was optimized to constructe a full-length enriched cDNA library of tobacco seedling leaves. The assembly and annotation of EST data were completed by bioinformatics program. 【Result】 A high quality full-length cDNA library was constructed successfully from tobacco leaves. The titer was 1.2×106 pfu•mL-1 and the average length of inserted cDNA fragments was 1.4 kb. A total of 5 280 clones were sequenced from the library, and 5 233 expressed sequence tag (EST) sequences were obtained. The ESTs were assembled into 3 922 unigenes including 564 contigs and 3 358 singletons. The comparative analysis shows that approximately 89.7% of the unigenes have homologs in other plants. These genes were involved in cell development, signal transduction, transcription and synthesis, stress tolerance response, energy metabolism and so on. Nicotine synthesis and transform, and disease resistance genes were identified. 【Conclusion】 A high-quality full-length cDNA library of tobacco leaves was successfully constructed by Cap-trapper method. Meanwhile, development, nicotine synthesis and transform, stress resistance and disease resistance were identified by sequences analysis of EST.

Key words: tobacco, full-length cDNA library, EST, function annotation

龚达平, 解敏敏, 孙玉合. 烟草叶片全长cDNA文库构建及EST序列分析[J]. 中国农业科学, 2012, 45(9): 1696-1702.

GONG Da-Ping, JIE Min-Min, SUN Yu-He. Construction and Sequences Analysis of Full-Length cDNA Library of Tobacco Leaves[J]. Scientia Agricultura Sinica, 2012, 45(9): 1696-1702.

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参考文献

[1]Bindler G, van der Hoeven R, Gunduz I, Plieske J, Ganal M, Rossi L, Gadani F, Donini P. A microsatellite marker based linkage map of tobacco. Theoretical and Applied Genetics, 2007, 114(2): 341-349.

[2]Bindler G, Plieske J, Bakaher N, Gunduz I, Ivanov N, van der Hoeven R, Ganal M, Donini P. A high density genetic map of tobacco (Nicotiana tabacum L.) obtained from large scale microsatellite marker development. Theoretical and Applied Genetics, 2011, 123(2): 219-230.

[3]Edwards K D, Bombarely A, Story G W, Allen F, Mueller L A, Coates S A, Jones L. TobEA: An atlas of tobacco gene expression from seed to senescence. BMC Genomics, 2010, 11: 142.

[4]European Sequencing of Tobacco Project [http://www.estobacco.info/]

[5]毛新国, 孔秀英, 赵光耀, 贾继增. 利用改进的Cap-trapper法构建拟斯卑尔脱山羊草(Aegilops speltoides Tausch)全长测DNA文库. 遗传学报, 2005, 32: 811-817.

Mao X G, Kong X Y, Zhao G Y, Jia J Z. Construction of a full-length cDNA library of Aegilops speltoides Tausch with optimized cap-trapper method. Acta Genetica Sinica, 2005, 32: 811-817. (in Chinese)

[6]Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, Itoh M, Kamiya M, Shibata K, Sasaki N, Izawa M, Muramatsu M, Hayashizaki Y, Schneider C. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics, 1996, 37: 327-336.

[7]Carninci P, Westover A, Nishiyama Y, Ohsumi T, Itoh M, Nagaoka S, Sasaki N, Okazaki Y, Muramatsu M, Schneider C, Hayashizaki Y. High efficiency selection of full-length cDNA by improved biotinylated cap trapper. DNA Research, 1997, 4: 61-66.

[8]Seki M, Carninci P, Nishiyama Y, Hayashizaki Y, Shinozaki K. High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper. The Plant Journal, 1998, 15: 707-720.

[9]Laboratory of Phil Green. http://www.phrap.org/

[10]Gordon D, Abanjian C, Green P. Consed: A graphical tool for sequence finishing. Genome Research, 1998, 8: 195-202.

[11]National Center for Biotechnology Information. http:∥www.ncbi.nlm. nih.gov

[12]Hunter S, Apweiler R, Attwood T K, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, Finn R D, Gough J, Haft D, Hulo N, Kahn D, Kelly E, Laugraud A, Letunic I, Lonsdale D, Lopez R, Madera M, Maslen J, McAnulla C, McDowall J, Mistry J, Mitchell A, Mulder N, Natale D, Orengo C, Quinn A F, Selengut J D, Sigrist C J, Thimma M, Thomas P D, Valentin F, Wilson D, Wu C H, Yeats C. InterPro: The integrative protein signature database. Nucleic Acids Research, 2009, 37: 211-215.

[13]Häkkinen S T, Tilleman S, Swiatek A, De Sutter V, Rischer H, Vanhoutte I, Van Onckelen H, Hilson P, Inzé D, Oksman-Caldentey K M, Goossens A. Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco. Phytochemistry, 2007, 68(22/24): 2773-2785.

[14]Siminszky B, Gavilano L, Bowen W S, Dewey E R. Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102: 14919-14924.

[15]Hulbert S H, Webb C A, Smith S M, Sun Q. Resistance gene complexes: Evolution and utilization. Annual Review of Phytopathology, 2001, 39: 285-312.

[16]Tobacco Genome Initiative. http://www.pngg.org/tgi

[17]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. Biotechniques, 2001, 30(4): 892-897.

[18]Suzuki Y, Yoshitomo Nakagawa K, Maruyama K, Suyama A, Sugano S. Construction and characterization of a full length enriched and a 5’-end-enriched cDNA library. Gene, 1997, 200(1/2): 149-156.

[19]Suzuki Y, Sugano S. Construction of a full length enriched and a 5’ end enriched cDNA library using the oligo capping method. Methods Molecular Biology, 2003, 221(1): 73-91.

[20]Edery I, C hu L L, Sonenberg N, Pelletier J. An efficient strategy to isolate full length cDNAs based on an mRNA cap retention procedure (CAPture). Molecular and Cellular Biology, 1995, 15(6): 3363-3371.

[21]Efmiov V A, Chakhmakhcheva O G, Archdeacon J, Fernandez J M, Fedorkin O N, Dorokhov Y L, Atabekov J G. Detection of the 5’ cap structure of messenger RNAs with the use of the cap jumping approach. Nucleic Acids Research, 2001, 29(22): 4751-4759.

[22]Seki M, Carninci P, NishiyamaY, Hayashizaki Y, Shinozaki K. High efficiency cloning Arabidopsis is full length cDNA by biotinylated CAP trapper. The Plant Journal, 1998, 15(5): 707-720.

[23]Carninci P, Shibata Y, Hayatsu N, Sugahara Y, Shibata K, Itoh M, Konno H, Okazaki Y, Muramatsu M, Hayashizaki Y. Normalization and subtraction of cap trapper selected cDNAs to prepare full length cDNA libraries for rapid discovery of new genes. Genome Research, 2000, 10(10): 1617-1630.

[24]赵光耀, 孔秀英, 贾继增. 粗山羊草（Ae.tauschii）幼苗和根全长cDNA文库构建及其EST注释与比较分析. 中国农业科学, 2007, 40(7): 1331-1336.

Zhao G Y, Kong X Y, Jia J Z. Full-length cDNA libraries construction from Ae. tauschii root and shoot and EST annotation and comparative analysis. Scientia Agricultura Sinica, 2007, 40(7): 1331-1336. (in Chinese)

[25]Whitham S, Dinesh-Kumar S P, Choi D, Hehl R, Corr C, Baker B. The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor. Cell, 1994, 78: 1101-1115.

[26]Hibi N, Higashiguchi S, Hashimoto T, Yamada Y. Gene expression in tobacco low-nicotine mutants. The Plant Cell, 1994, 6: 723-735.