苹果木葡聚糖酶抑制蛋白基因的克隆与表达分析

Molecular cloning and expression analysis of XEGIP in Malus pumila

以‘富士’苹果(Malus pumila)树皮总RNA为模版,利用RT-PCR技术克隆木葡聚糖酶抑制蛋白基因,命名为Mp XEGIP1。通过生物信息学软件对基因c DNA序列、系统进化关系及理化性质进行分析,采用荧光定量PCR技术测定Mp XEGIP1的表达量。构建原核表达载体p ET-Mp XEGIP1,转化大肠杆菌Rosetta(DE3),利用SDSPAGE和western blot检测和验证融合蛋白的表达。结果表明,获得了Mp XEGIP1的全长c DNA序列(登录号MG515243),开放阅读框1 344 bp,编码447个氨基酸。Mp XEGIP1编码蛋白的理论分子量约48.77 k Da,为亲水的不稳定蛋白;57~419位氨基酸为木聚糖酶抑制蛋白保守域。序列多重比对及系统进化树分析表明,Mp XEGIP1编码氨基酸序列与‘金冠’苹果XEGIP完全一致,其次与梨XEGIP序列相似性81%,三者亲缘关系较近。‘富士’枝条接种腐烂病菌后,Mp XEGIP1显著上调表达。原核表达和western blot分析表明,该蛋白以包涵体形式存在,能与HIS抗体特异性结合,证实Mp XEGIP1编码蛋白得到成功表达。

Xyloglucan-specific endo-β-1,4-glucanase inhibitor protein（XEGIP） gene was amplified by RT-PCR from barks of Malus pumila ‘Fuji＇, named MpXEGIP1. The full-length cDNA sequence, phylogenetic relationship and physicochemical properties of this gene were analyzed by a variety of bioinformatics software. The expression levels of MpXEGIP1 were analyzed by real-time quantitative PCR（q PCR）. Then, a recombinant plasmid p ET-MpXEGIP1 was constructed and transformed into Escherichia coli Rosetta（DE3）. Recombinant protein expression and solubility of MpXEGIP1 were detected and verified by SDS-PAGE and western blot. The results showed that the full-length cDNA sequence of MpXEGIP1 from M. pumila was obtained and its Gen Bank accession number was MG515243. The open reading frame（ORF） of 1 344 bp encodes 447 amino acid residues with a predicted molecular weight of 48.77 kDa. MpXEGIP1 is an unstable hydrophilic protein and has a conserved domain of xylanase inhibitor at the amino acid residues from 57 to 419. Multiple sequence alignment and phylogenetic tree analysis showed that the encoded protein had the closest genetic relationship with M. pumila ‘Golden Delicious＇ and Pyrus bretschneideri XEGIP and the similarities of amino acid sequences were 100% and 81%, respectively. q PCR results showed that the expression of MpXEGIP1 was significantly up-regulated in apple twigs after inoculation by Valsa mali. SDS-PAGE and western blot results showed that the fused protein mainly existed in the form of inclusion body, and the protein could be specifically recognized by the HIS monoclonal antibody, which further confirmed that MpXEGIP1 protein was successfully expressed.