Cloning, expression, and bioinformatics analysis of the chitinase gene PlCHI in Picea likiangensis var. balfouriana
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摘要: 以川西云杉（Picea likiangensis var.balfouriana（Rehd.et Wils.）Hillier ex Slsvin）为材料，利用RT-PCR技术，克隆获得几丁质酶基因PlCHI的cDNA全长序列，并对该基因的序列特征及基因表达情况进行分析。结果显示，PlCHI的开放阅读框长1017 bp，共编码338个氨基酸；蛋白序列结构域分析结果表明，PlCHI为ClassⅠ类几丁质酶，属19家族，兼具溶菌酶活性；该序列与其他植物几丁质酶的蛋白序列相似性较高。系统发育分析结果显示，川西云杉PlCHI序列与北美云杉（Picea sitchensis（Bong.）Corr.）、黑松（Pinus thunbergii Parl.）等松科植物亲缘关系最近。进一步将PlCHI重组质粒转入大肠杆菌BL21（DE3）中，表达出约45 kD的蛋白，主要以包涵体形式存在，且在25℃下采用0.2 mmol/L的IPTG诱导4 h时蛋白的表达量最佳。Abstract: The full-length cDNA of the chitinase gene PlCHI was cloned from Picea likiangensis var. balfouriana (Rehd. et Wils.) Hillier ex Slsvin by RT-PCR, with sequence characteristics and gene expression levels then analyzed. Results showed that the open reading frame of PlCHI was 1017 bp in length and encoded a protein with 338 amino acids. Protein sequence domain analysis indicated that PlCHI was a class Ⅰ chitinase that belonged to the 19 family and exhibited lysozyme activity. The sequence of PlCHI had high similarity to the protein sequences of other plant chitinases. Phylogenetic analysis showed that the PlCHI sequence was closely related to Pinaceae plants such as Picea sitchensis (Bong.) Corr. and Pinus thunbergii Parl. In this study, we further transferred the PlCHI recombinant plasmid into E. coli BL21 (DE3) to express a protein of about 45 kD, which was mainly in the form of an inclusion body. The optimal expression conditions of the gene were 0.2 mmol/L IPTG at 25℃ for 4 h induction.
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