桑树几丁质酶基因Machi1的克隆及原核表达

Cloning of Chitinase Gene Machi1 in Mulberry and Its Prokaryotic Expression

以桑品种云桑2号的健康叶片为材料,通过反转录PCR结合RACE方法克隆到桑树几丁质酶基因Machi1(GenBank登录号:MK783295)。Machi1基因的完整ORF序列长度为972 bp,编码323个氨基酸残基,预测Machi1蛋白分子质量为349kD,理论等电点(pI)为684。结构分析显示Machi1蛋白含有一个信号肽,无跨膜结构域。Machi1蛋白属于糖苷水解酶19家族,含有保守的ChtBD1结构域,预测具有几丁质酶活性。多序列比对结果表明,Machi1蛋白与川桑几丁质酶Mnchi19一致性最高为935%,系统进化树结果与之相一致。组织表达模式分析结果表明,Machi1基因在桑树叶片中的表达量最高。随后通过原核表达系统获得了重组目的蛋白并进行了纯化,用纯化的蛋白质免疫新西兰大白兔制备Anti-Machi1多克隆抗体。利用间接ELISA法测定其效价在1∶80 000以上,并经Western blotting验证了该多克隆抗体的特异性,为后续研究Machi1的抗病机制及生物学功能奠定了基础。

Mulberry chitinase gene Machi1(GenBank accession No.:MK783295)was cloned from healthy leaves of mulberry variety Yunsang 2 by reverse transcription PCR combined with RACE technique. The open reading frame(ORF)of Machi1 gene is 972 bp and encodes a polypeptide of 324 amino acids with predicted molecular weight of 34 9 kD and isoelectric point(pI)of 6 84. Structural analysis revealed that Machi1 protein contains a signal peptide and has no transmembrane domain. As a member of glycoside hydrolase 19 family,Machi1 protein contains a conserved chitin binding domain(ChtBD1),so it was predicted to have chitinase activity. Multiple sequence alignment showed that Machi1 amino acids shared 93 5% similarity with Mnchi19 of Morus notabilis,which was consistent with the phylogenetic analysis. Tissue expression pattern analysis showed that expression level of Machi1 gene was the highest in mulberry leaf. Furthermore,the recombinant protein was obtained and purified by prokaryotic expression,and then used to immunize rabbit to prepare polyclonal antibody Anti-Machi1. It was testified by indirect ELISA assay that the titer of AntiMachi1 was above 1 ∶ 80 000,and specificity of this polyclonal antibody was verified by Western blotting. This study laid a foundation for the subsequent study on disease resistance mechanism and biological function of Machi1.