Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis

羊草Class Ⅱ几丁质酶基因的克隆及序列分析

[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.

1材料与方法1．1植物材料采摘自然生长于黑龙江省安达市（119°28’E，44°1’N）盐碱地的羊草叶片，-70℃冰箱保存。1．2总RNA的分离与eDNA文库的构建用液氮研磨叶片后，用TRIZOL Reagent（GIBCO BRL）提取总RNA，使用PolyATtract mRNA Isolation Systems（Promega）提取mRNA。然后采用Stratagene提供的cDNA合成体系合成cDNA，体外包装和扩增，构建羊草叶片的cDNA文库。