Construction of a BAC library of Physcomitrella patens and isolation of a LEA gene
Construction of a BAC library of Physcomitrella patens and isolation of a LEA gene
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DOI：
10.1016/j.plantsci.2004.04.015
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发表时间：
2004-09-01
期刊：
影响因子：
5.2
通讯作者：
Wang, B
中科院分区：
文献类型：
--
作者：
Liang, CY;Xi, Y;Wang, B
小立碗藓是研究植物生理和发育生物学的理想模式系统。藓类植物的进化地位使其成为研究陆生植物进化的理想对象。此外，由于展幅杨具有较强的耐旱、耐盐、耐寒性，可能含有重要的抗病基因资源。基于这些原因，构建了一个Ppatens的细菌人工染色体（BAC）文库。用HindIII部分消化高分子量（HMW）DNA。通过脉冲场凝胶电泳（PFGE）进行一次大小分级，并将选择的片段连接到载体pGUGIBAC 1。将连接混合物转化到DH 10 B细胞中并挑取重组体。总共收集了49，920个BAC克隆。BAC文库的平均插入片段大小为65 kb，基因组覆盖率约为7倍。叶绿体和线粒体基因的杂交结果表明，BAC文库没有明显的细胞器DNA污染。BAC克隆使用两种方法储存：一种涉及将文库储存在384孔板中，1个克隆在1个孔中;另一种是将文库储存在96孔板中，12个克隆合并在一起进入孔中。晚期胚胎发生丰富（莱亚）蛋白被认为是响应脱水、渗透和低温胁迫的保护性成分。以莱亚基因为探针，采用快速PCR方法对BAC文库进行筛选，筛选出6个阳性克隆。所构建的BAC文库将为P patens的基因筛选和克隆奠定基础。此外，该BAC文库的构建为开展小帕氏杆菌的基因组研究提供了一个有价值的工具。(C)2004爱思唯尔爱尔兰有限公司保留所有权利。
The moss Physcomitrella patens is an ideal model system for the study of plant physiology and developmental biology. The evolutionary position of moss makes P. patens an ideal subject for the study of land plant evolution. Furthermore, as P. patens is highly tolerant to drought, salt and freezing, it may contain important resistant gene resources. For these reasons, a bacterial artificial chromosome (BAC) library of P patens was constructed. The high molecular weight (HMW) DNA was partially digested with HindIII. One size fractionation was carried out by pulsed field gel electrophoresis (PFGE) and the selected fragments were ligated to the vector pGUGIBAC1. The ligation mixture was transformed into DH10B cells and recombinants were picked out. In total, 49,920 BAC clones were collected. The BAC library has an average insert size of 65 kb and about seven-fold genome coverage. The hybridization results probed by chloroplast and mitochondrial genes demonstrated that the BAC library has no significant organellar DNA contaminations. The BAC clones were stored using two methods: one involved storing the library in 384-well plates, 1 clone in 1 well; in the other, the library was stored in 96-well plates, 12 clones being pooled together into well. Late embryogenesis-abundant (LEA) proteins are thought to act as a protective component in respond to dehydration, osmotic, and low-temperature stresses. The BAC library was screened with a LEA gene as a probe using rapid PCR methods and six positive clones were screened Out. The constructed BAC library will be useful for gene screening and cloning of P patens. In addition, this BAC library provides a valuable tool for the genomic study of P patens. (C) 2004 Elsevier Ireland Ltd. All rights reserved.