云南松基因组微卫星富集文库的构建

生物技术通报 ›› 2014, Vol. 0 ›› Issue (1): 93-99.

云南松基因组微卫星富集文库的构建

许玉兰^1,2,张瑞丽²,田斌²,蔡年辉²,康向阳¹,段安安²

1. 北京林业大学林木育种国家工程实验室，北京 100083#br# 2. 西南林业大学国家林业局西南地区生物多样性保育重点实验室，昆明 650224

收稿日期:2013-07-17 出版日期:2014-01-23 发布日期:2014-01-23
作者简介:许玉兰，女，副教授，博士研究生，研究方向: 林木遗传育种的研究；E-mail: xvyulan@163.com
基金资助:
国家自然科学基金项目(31260191)，云南省自然科学基金项目(2010CD065)，云南省省院省校教育合作咨询共建重点学科(211015)，云南省教育厅研究生项目(2012J052)

Construction of Microsatellite-enriched Library of Pinus yunnanensis

Xu Yulan^1,2, Zhang Ruili², Tian Bin², Cai Nianhui², Kang Xiangyang¹, Duan Anan²

1. College of Biological Science and Technology of Beijing Forestry University, National Engineering Laboratory for Tree Breeding，Beijing Forestry University，Beijing 100083; #br# 2. Key Laboratory of Biodiversity Conservation in Southwest China, State Forestry Administration， Southwest Forestry University，Kunming 650204

Received:2013-07-17 Published:2014-01-23 Online:2014-01-23

摘要/Abstract

摘要： 采用磁珠富集法构建云南松微卫星富集文库。云南松基因组DNA 经Rsa Ⅰ酶切，与特定接头连接，再用接头特异#br# 引物进行PCR 扩增。连接扩增产物与用生物素标记的(AG)12、(AT)12、(CG)12、(GT)12、(ACG)12、(ACT)12 和(CCA)8#br# 探针杂交，通过链霉亲和素偶联的磁珠捕捉含接头和微卫星序列的片段并扩增，将获得的片段连接到pMD-19T 载体上，转化至大#br# 肠杆菌JM109 感受态细胞中，成功构建了云南松微卫星富集文库。通过PCR 检测从文库中筛选阳性克隆，在383 富集阳性菌落中#br# 获得阳性克隆257 个，经测序分析，在获得的159 条序列中，有143 条含有SSR，其中完美型占65.73%，非完美型占23.78%，混#br# 合型占10.49% 。结果表明，磁珠富集法构建云南松基因组微卫星文库高效、可行的，文库的构建为微卫星位点的分离、遗传多样#br# 性的分析等奠定基础。

Abstract: Microsatellite-enriched libraries in Pinus yunnanensis were constructed using an enrichment method with biotin-labeled probes and streptavidin-coated magnetic beads.Genomic DNAs of P. yunnanensis were extracted and digested with restriction enzyme Rsa 1. Adapters were ligated to the end of restriction fragments. Ligated products were amplified by PCR with primers complementary to adapters. The PCR products were hybridized with biotin-labeled simple sequence repeat probes(AG) (AT) (CG) (GT)12(ACG) , (ACT )12 and(CCA) 12, 12, 12, 12,8. The hybridized complex was caught by magnetic beads coated with streptavidin. The biotinylated hybrids were isolated on the magnetic particles according to the strong affinity between biotin and streptavidin. The selected DNAs were amplified by PCR with primers complementary to adapters and cloned into pMD-19T plasmid vector, and then transformed into E. coli JM109 hosts. SSR enriched genomic DNA library of P. yunnanensis was constructed. A total of 257 positive clones were selected by PCR from 383 transformants in the microsatellite-enriched library,and 143 clones were found to contain simple sequence repeats(SSR)among 159 sequences. Among them, 94(65.73%)were perfect repeat,34 imperfect(23.78%)and 15(10.49%)compound repeat. The results indicated that the method was efficient to construct the microsatelliteenriched library of P. yunnanensis. The library will be useful for further research on the isolation of microsatellite markers and the analysis of genetic diversity..

许玉兰,张瑞丽,田斌,蔡年辉,康向阳,段安安. 云南松基因组微卫星富集文库的构建[J]. 生物技术通报, 2014, 0(1): 93-99.

Xu Yulan, Zhang Ruili, Tian Bin, Cai Nianhui, Kang Xiangyang, Duan Anan. Construction of Microsatellite-enriched Library of Pinus yunnanensis[J]. Biotechnology Bulletin, 2014, 0(1): 93-99.

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