橡胶树甲基化过滤文库的构建

doi:10.3724/SP.J.1005.2010.01071

遗传 ›› 2010, Vol. 32 ›› Issue (10): 1071-1076.doi: 10.3724/SP.J.1005.2010.01071

橡胶树甲基化过滤文库的构建

黄关青, 华玉伟, 高战, 黄华孙

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 出版日期:2010-10-20 发布日期:2010-10-20
通讯作者: 黄华孙 E-mail:xjshhs@163.com
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

HUANG Guan-Qing, HUA Yu-Wei, GAO Zhan, HUANG Hua-Sun

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:2010-01-20 Revised:2010-05-19 Online:2010-10-20 Published:2010-10-20
Contact: HUANG Hua-Sun E-mail:xjshhs@163.com

摘要/Abstract

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。

关键词: 甲基化过滤, 基因富集, 文库构建, 巴西橡胶树

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.

Key words: Hevea brasiliensis, methylation filtration, gene enrichment, library construction

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.

HUANG Guan-Jing, HUA Yu-Wei, GAO Zhan, HUANG Hua-Sun. Construction of a methylation filtration library in Hevea brasiliensis[J]. HEREDITAS, 2010, 32(10): 1071-1076.

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橡胶树甲基化过滤文库的构建

Construction of a methylation filtration library in Hevea brasiliensis

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