梨几丁质酶基因克隆及其生物信息学分析

Cloning of Pear Chitinase Gene and Bioinformatics Analysis

对克隆获得的梨几丁质酶基因Lchi1进行生物信息学分析,从而为该基因的功能鉴定提供理论基础,以期为梨的抗病育种提供基因来源。根据Genbank中已克隆得到的几丁质酶基因保守区,在梨基因组数据库中进行Blast比对,调取DNA序列,从而设计引物,以高抗腐烂病品种‘小香水’(Pyrus ussuriensis cv.Xiaoxiangshui)为研究材料,克隆获得梨几丁质酶Lchi1基因c DNA全长,并对其进行初步的生物信息学分析。梨基因组数据库中调取的DNA序列2535 bp,含有3个外显子区、2个内含子区。Lchi1基因全长1300 bp,编码322个氨基酸,其蛋白的分子式为C1607H2407N415O468S17,分子量为35573.3 Da,理论等电点为7.10。二级结构以无规则卷曲和α-螺旋为主,三级结构具有高度保守性。Lchi1编码蛋白为可溶性胞外蛋白,存在少量的潜在磷酸化位点,属于糖苷水解酶19家族,含有N-端信号肽,但是缺乏几丁质结合区。Lchi1基因属于CLASSⅡ几丁质酶基因,与其他科属植物的几丁质酶基因相似性均较高。

The pear chitinase gene Lchi1 was cloned and the gene related information of the bioinformatics wasanalyzed. Thus the study could provide a theoretical basis for the identification of gene function, and provide asource of gene for pear resistance breeding. According to the conserved sequence of the plant chitinase gene inGenbank, Blast was carried out in the pear genome database, and the DNA sequence was retrieved. Primerswere designed according to the DNA sequence. c DNA fragments of chitinase gene was amplified from‘XiaoXiang Shui＇（Pyrus ussuriensis cv. Xiaoxiangshui）. Bioinformatics analysis was also conducted. The DNAsequence was 2535 bp, including three exon regions and two intron regions. Bioinformatics analysis showedthat the full length of Lchi1 gene was 1300 bp and it contained a complete ORF which encoded 322 aminoacids. The predicted protein＇s molecular formula was C1607H2407N415O468S17, whose relative molecular mass was35573.3 Da and isoelectric point was 7.10. The secondary structure of the protein mainly included random coiland α- helix. Tertiary structure was highly conservative. It was predicted that the protein was soluble and extracellular protein, and there were a small amount of potential phosphorylation sites. The protein belonged toglycoside hydrolase 19 family, and there was a signal peptide and no chitin binding domain. Lchi1 genebelonged to CLASS Ⅱ chitinase gene, which was highly conservative among chitinases from other differentplant species.