摘要： 为探究苹果树腐烂病菌β-葡萄糖苷酶基因VmGluI的序列特征及其在病菌侵染致病过程中的表达，以强致病力菌株LXS080601为材料，利用RT-PCR技术克隆基因cDNA序列，通过生物信息学软件对VmGluI编码蛋白的系统进化关系、理化性质和二级结构进行分析，采用实时荧光定量PCR技术测定VmGluI的表达量。结果显示，苹果树腐烂病菌VmGluI的开放阅读框为1 689 bp（GenBank登录号KY646110），编码562个氨基酸，蛋白分子量62.7 kDa。VmGluI编码的蛋白具有糖基水解酶1家族的保守功能域，其二级结构主要以α-螺旋和无规则卷曲为主，β-转角较少。VmGluI编码氨基酸序列与苹果和梨树腐烂病菌β-葡萄糖苷酶基因（KUI68239.1和KUI56905.1）同源性最高，分别为98.7%和87.8%，而与非植物病原真菌β-葡萄糖苷酶基因亲缘关系较远。接种苹果树腐烂病菌的富士枝条中，VmGluI显著上调表达，推测其在腐烂病菌侵染致病过程中起重要作用。离体枝条接种48 h后基因表达量开始显著升高，72 h时表达量最高为对照6.3倍；活体枝条接种12 h后，基因表达量已为对照的11.2倍，接种后48 h基因表达出现低谷，但96 h时表达量为对照的16.8倍，表明苹果树腐烂病菌在侵染离体和活体枝条过程中，VmGluI表达水平和时序变化存在明显差异。

关键词: 苹果树腐烂病菌, β-葡萄糖苷酶基因, 基因克隆, 生物信息学分析, 基因表达

Abstract: To explore the sequence characteristics of β-glucosidase gene VmGluI from Valsa mali var. mali (Vmm)and analyze its expression pattern in the process of pathogen infection,the cDNA sequence of VmGluI was amplified from the strong pathogenic strain LXS080601 by using RT-PCR technology.The phylogenetic relationship,physicochemical properties and secondary structure of the protein coded by VmGluI were analyzed by bioinformatics software.The expression levels of VmGluI in apple tiwgs(Malus domestica Borkh.Fuji) inoculated by Vmm were analyzed by Real-time quantitative PCR.The results showed that the cDNA sequence of VmGluI from Vmm was obtained and its GenBank accession number was KY646110.The open reading frame of 1 689 bp encoded 562 amino acid residues with a predicted molecular weight of 62.7 kDa.VmGluI shared characteristic conserved domains of glycosyl hydrolase family 1. α -helices and random coils were its main elements of secondary structure of VmGluI,and β-turns were less.Multiple sequence alignment and phylogenetic tree analysis showed that the encoded protein had the closest genetic relationship with β-glucosidase genes from Vmm (KUI68239.1) and V.mali var. pyri (KUI56905.1),and the similarities of amino acid sequences were 98.7% and 87.8%,respectively.However,the relationship between VmGluI and other β-glucosidase genes from non-pathogenic fungi was far away.The Real-time quantitative PCR results showed that VmGluI was significantly up-regulated in Fuji tiwgs after inoculation by Vmm.Thus,the authors speculated that VmGluI could paly an important role in the pathogenic process of Vmm.The expression level of VmGluI began to increase significantly in detached twigs at 48 h and the highest expression level was 6.3 times higher than that in the control at 72 h.However,the gene expression level increased significantly at 12 h in living twigs,which was 11.2 times higher than that in the control at 96 h.Although there was a low level of gene expression at 48 h,the amount of gene expression was 16.8 times higher than that in the control at 96 h.The results indicated that expression levels and the time course changes of VmGluI were obvious difference in detached and living twigs inoculated with Vmm.

Key words: Valsa mali var.mali, VmGluI, Gene cloning, Bioinformatics analysis, Gene expression

中图分类号: 

• Q78

• S436.03