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橡胶树甲基化过滤文库的构建

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  • 中国热带农业科学院橡胶研究所, 农业部橡胶树生物学重点开放实验室, 国家橡胶树育种中心, 海南儋州 571737

收稿日期: 2010-01-20

  修回日期: 2010-05-19

  网络出版日期: 2010-10-20

基金资助

国家天然橡胶产业技术体系项目(编号:nycytx-34)和农业部农作物种质资源保护项目(编号:NB09-2130135-14)资助

Construction of a methylation filtration library in Hevea brasiliensis

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  • State Center for Rubber Breeding, Key Laboratory of Rubber Biology of Ministry of Agriculture, Rubber Research Institute of Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, China

Received date: 2010-01-20

  Revised date: 2010-05-19

  Online published: 2010-10-20

摘要

为高通量富集橡胶树基因编码序列, 文章首次利用大肠杆菌McrBC限制修饰系统构建了巴西橡胶树基因组甲基化过滤文库。该文库未扩增和扩增后的滴度分别为2.6×106 pfu/mL和9×109 pfu/mL, 阳性克隆率为86.4%, 插入片段大小为1~2.5 kb, 平均长度为1.2 kb。随机挑选100个克隆进行测序, 共拼接出81个非冗余序列, 其中包括6个重叠群(Contigs)和75个单一序列(Singlets), 冗余度为17.35%。Blast分析发现, 39.5%非冗余序列与Nr库中功能基因同源, 14.81%与EST数据库中序列同源, 32.1%序列为未知序列, 且部分序列为开花调控、抗虫和抗病等功能相关的同源基因。橡胶树甲基化文库的建立为橡胶树重要功能基因发现和克隆提供一条重要途径。

本文引用格式

黄关青,华玉伟,高战,黄华孙 . 橡胶树甲基化过滤文库的构建[J]. 遗传, 2010 , 32(10) : 1071 -1076 . DOI: 10.3724/SP.J.1005.2010.01071

Abstract

In order to enrich gene encoding region of Hevea brasiliensis, a methylation filtration library was constructed using Escherichia coli McrBC restriction-modification system. The titers of the non-amplified library and the amplified library were 2.6×106 pfu/ml and 9.0×109, respectively. The rate of positive clones was 86.4%. The lengths of inserted DNA sequence ranged from 1 kb to 2.5 kb and the average size of inserts was 1.2 kb. One hundred clones were selected randomly for sequencing, resulting in splicing out of 81 non-redundant sequences, including 6 contigs and 75 singlets. The redun-dancy was 17.35%. Blast analysis showed that 39.5% of non-redundant sequences were homologous with the Nr database, 14.81% with the EST database, and 32.1% were unknown sequences. Some sequences were related genes for flowering, insect and disease resistance. Therefore, the rubber tree methylation library is helpful for discovery and cloning of func-tional genes.

参考文献

[1] 程汉, 蔡海滨, 黄华孙. 巴西橡胶树低温诱导全长cDNA文库构建. 热带作物学报, 2008, 29(4): 410–414. [2] Miao Z, Gaynor JJ. Molecular cloning, characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis). J Mol Biol, 1993, 23(2): 267–277. [3] Chey ML, Kush A, Tan CT. Characterization of cDNA and genomic clones encoding HMGCoA reductase from Hevea brasilienis. Plant Mol Biol, 1991, 16: 567. [4] 华玉伟, 卢轶, 龙青姨, 曾霞, 胡彦师, 黄华孙. 橡胶树EST-SSRs发掘研究. 热带作物学报, 2008, 29(5): 583–588. [5] Lespinasse D, Rodier GM, Grivet L, Leconte A, Legnate H, Seguin M. A saturated genetic linkage map of rubber tree (Hevea spp.) based on RFLP, AFLP, microsatellite, and isozyme markers. Theor Appl Genet, 2000, 100: 127–138. [6] Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR. Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell, 2006, 126(6): 1025–1038. [7] Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, Zhang X, Bernstein BE, Nusbaum C, Jaffe DB, Gnirke A, Jaenisch R, Lander ES. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature, 2008, 454(7): 766–771. [8] Rabinowicz PD, Schutz K, Dedhia N, Yordan C, Parnell LD, Stein L, McCombie WR, Martienssen RA. Differen-tial methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nat Genet, 1999, 23(3): 305–308. [9] Bedell JA, Budiman MA, Nunberg A, Citek RW, Robbins D, Jones J, Flick E, Rholfing T, Fries J, Bradford K, McMenamy J, Smith M, Holeman H, Roe BA, Wiley G, Korf IF, Rabinowicz PD, Lakey N, McCombie WR, Jed-deloh JA, Martienssen RA. Sorghum genome sequencing by methylation filtration. PLoS Biol, 2005, 3(1): e13. [10] Timko MP, Rushton PJ, Laudeman TW, Bokowiec MT, Chipumuro E, Cheung F, Town CD, Chen X. Sequencing and analysis of the gene-rich space of cowpea. BMC Genomics, 2008, 9: 103. [11] Seguin M, Flori A, Legnaté H, Clément-Demange A. Rubber tree. In: Hamon P, Seguin M, Perrier X, Glasz-mann JC, eds. Genetic Diversity of Tropical Crops. Cirad, France and Science Publ., Enfield, USA, 2003, 277–306. [12] Low FC, Bonner J. 1985. Characterization of the nuclear genome of Hevea brasiliensis. Proc. Int. Rubb. Conf. Kuala Lumpur, Malaysia, 1–9. [13] 安泽伟, 黄华孙. 一种提取橡胶树叶中总DNA的方法. 植物生理学通讯, 2005, 41(4): 513–515. [14] Hanley S, Edwards D, Stevenson D, Haines S, Hegarty M, Schuch W, Edwards KJ. Identification of transpo-son-tagged genes by the random sequencing of Muta-tor-tagged DNA fragments from Zea mays. Plant J, 2000, 23(4): 557–566. [15] Cowperthwaite M, Park W, Xu Z, Yan X, Maurais SC, Dooner HK. Use of the transposon Ac as a gene-searching engine in the maize genome. Plant Cell, 2002, 14(3): 713–726. [16] Fernandes J, Dong QF, Schneider B, Morrow DJ, Nan GL, Brendel V, Walbot V. Genome-wide mutagenesis of Zea mays L. using RescueMu transposons. Genome Biol, 2004, 5: R82. [17] Peterson DG, Schulze SR, Sciara EB, Lee SA, Bowers JE, Nagel A, Jiang N, Tibbitts DC, Wessler SR, Paterson AH. Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome charac-terization and gene discovery. Genome Res, 2002, 12(5): 795–807. [18] Yuan YN, SanMiguel PJ, Bennetzen JL. High-Cot se-quence analysis of the maize genome. Plant J, 2003, 34(2): 249–255. [19] Lamoureux D, Peterson DG, Li W, Fellers JP, Gill BS. The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.). Genome, 2005, 48(6): 1120–1126. [20] Adams MD, Kelley JM, Gocayne JD, Dubnick M, Poly-meropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF. Complementary DNA sequencing: expressed sequence tags and human genome project. Science,
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