Construction of a BAC library of Physcomitrella patens and isolation of a LEA gene

Abstract

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 1 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.