苹果甘露糖结合蛋白2基因(MdMBP2)的克隆及其重组蛋白的活性研究  

Cloning of a Mannose-binding Protein 2 Gene(MdMBP2) from Malus domestica and Identification of Its Biological Activity

作　　者：柏素花[1,2,3] 李保华[4] 戴洪义[2] 

机构地区：[1]湖南农业大学园艺园林学院,长沙410128 [2]青岛农业大学园林园艺学院,青岛266109 [3]岛农业大学生命科学学院山东省高校植物生物技术重点实验室,青岛266109 [4]青岛农业大学农学与植物保护学院,青岛266109

出　　处：《农业生物技术学报》2013年第3期282-291,共10页Journal of Agricultural Biotechnology

基　　金：国家苹果产业技术体系项目(No.CARS-28-01-07);山东省良种产业化工程项目(No.620902);青岛市科技计划基础研究项目(No.12-1-4-5-(1)-jch)

摘　　要：甘露糖结合蛋白(MBP)是一类包含B-lectin结构域的植物凝集素,通过与糖的结合参与多种生理过程,包括细胞和细胞之间的相互作用以及寄主和病原之间的相互作用等。为了探讨苹果甘露糖结合蛋白在抗轮纹病防御反应中的作用,本研究从苹果(Malus demestica)枝条韧皮部cDNA鉴定了一个甘露糖结合蛋白的编码基因,命名为MdMBP2(GenBank accession No.JX126857)。该基因全长1553bp,开放阅读框为1368bp,编码一个由455个氨基酸残基组成的蛋白质,分子量和等电点分别为50.4kD和8.68。该蛋白的氨基酸序列包含保守的B-lectin结构域,N端有27个氨基酸残基组成的信号肽,C端有一个PAN-apple结构域。同源性和系统演化分析显示,MdMBP2蛋白与葡萄(Vitis vinifera)和大豆(Glycinemax)的表皮分泌蛋白(EP)及拟南芥(Arabidopsis thaliana)的甘露糖结合蛋白具有较高的序列相似性。包含B-lectin结构域的蛋白可以分成两个群,MdMBP2属于ClassⅠ,与ClassⅡ蛋白相比,ClassⅠ缺少SLP结构域和蛋白激酶结构域。利用荧光定量PCR技术检测MdMBP2基因的表达,发现MdMBP2基因在被检测的几种组织中均有不同程度的表达,但在皮和叶中的表达量较高,这种表达模式表明,MdMBP2可能参与多种生理途径。另外,轮纹病病原菌(Botryosphaeria dothidea)侵染能诱导MdMBP2转录本的积累,且其时序表达变化与轮纹病发病过程相关,暗示该基因参与了苹果抗轮纹病的防御反应。通过体外重组表达MdMBP2基因的重组蛋白,并用重组蛋白进行糖结合实验,发现MdMBP2蛋白能与真菌细胞壁组分高甘露糖型聚糖和甘露寡糖有较高的结合活性,表明MdMBP2蛋白可能参与病原真菌的识别。本研究分离鉴定了一个新的苹果甘露糖结合蛋白的编码基因,认为该基因参与苹果对轮纹病病原的防御反应,可能在寄主对病原的识别过程中发挥作用。Mannose binding protein （MBP） is a subgroup of plant lectin and thought to play a role in various biological processes such as cell-to-cell and host-pathogen interactions. In our previous experiments, an apple MBP gene was found among the highly expressed genes induced by inoculation with Botryosphaeria dothidea, a causal agent of a disastrous disease, apple ring rot. To investigate the role of the MBP gene in the defense responses ofapple（Ma/us domestica） against B. dothidea, we cloned the full-length cDNA sequence of the MBP gene and designated as MdMBP2 （GenBank accession No. JX126857）. The full-length cDNA was of 1 553 bp with a 1 368 bp of open read frame （ORF） encoding a protein of 455 amino acid residues. The calculated molecular weight and isoelectric point of the MdMBP2 protein were 50.4 kD and 8.68, respectively. Sequence and structure analysis indicated that MdMBP2 possessed a B-lectin domain and PAN-apple domain. There was a predicted signal peptide of 27 amino acids in the N terminus. BLAST analysis revealed that MdMBP2 had high identity to epidermis-specific secreted glycoprotein from Vitis vinifera （66%） and Glycine max （42%）, and mannose binding protein from A rabidopsis thaliana （57%）. Phylogenetic analysis revealed that all B-lectin domain containing proteins could be grouped into two classes and MdMBP2 belonged to the Class I which lacked SLP domain and PKc （protein kinase） domain compared with the proteins in Class lI. The expression of MdMBP2 gene was observed in all tissues examined here, and the highest expression of MdMBP2 gene was found in bark and leaf, and the lowest in root. This expression pattern suggested that MdMBP2 might be involved in many physiological pathways. The responses of MdMBP2 expression to B. dothidea infection were also examined using quantitative Real-time PCR. The results indicated that MdMBP2 expression was significantly enhanced by B. dothidea infection, and the temporal expression was related with the course of disease, which sugge

关 键 词：甘露糖结合蛋白 苹果 轮纹病菌 重组蛋白 糖结合活性 

分 类 号：Q513[生物学—生物化学]