长柄链格孢AlCyP1基因的克隆及其在渗透胁迫适应中的作用  

Cloning of AlCyP1 gene from Alternaria longipes and its functions in adaptation to osmotic stress

作　　者：罗义勇[1,2] 祝明亮[3] 路则宝 毕薇[1] 张克勤[1] 杨金奎[1] 

机构地区：[1]云南大学生物资源保护与利用国家重点实验室培育基地/教育部微生物资源重点实验室,云南昆明650091 [2]昆明理工大学生物工程技术研究中心,云南昆明650224 [3]云南省烟草科学研究所,云南玉溪653100 [4]楚雄医药高等专科学校医学检验系,云南楚雄675005

出　　处：《浙江大学学报（农业与生命科学版）》2011年第2期133-141,共9页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家重点基础研究发展计划资助项目(2007CB411600);云南省烟草公司科技资助项目(04A19);云南省中青年学术和技术带头人后备人才资助项目(2009CI052);云南省应用基础研究自筹经费资助项目(2010ZC056)

摘　　要：为了阐明烟草赤星病（tobaccobrown-spotdisease）病原真菌长柄链格孢（Alternarialongipes）对二甲酰亚胺类杀菌剂抗性的分子机制，以不同抗性水平的长柄链格孢菌株为实验材料，通过基因钓鱼（GeneFishing）技术进行基因差异表达分析；并利用基因敲除技术对已克隆到的AlCyP1基因进行功能分析．结果表明：一个亲环蛋白基因AlCyP1的表达量在不同抗性水平的长柄链格孢中存在明显差异；应用DNA步移（DNAwalking）方法克隆得到AlCyP1基因的DNA序列，其开放阅读框中存在2个翻译起始密码子，可以编码产生2种不同长度的多肽，长、短多肽产物分别具有222和188个氨基酸，其中短多肽起始于长多肽的第35个密码子；另外，氨基酸序列同源性分析发现，AlCyP1和其他真菌的亲环蛋白具有很高的同源性，达50．0％～61.3％；利用基因敲除技术对AlCyP1基因进行功能分析发现，该基因通过表达水平的改变参与了长柄链格孢对渗透胁迫的适应调节过程．In order to clarify the molecular mechanism of tobacco brown-spot disease pathogenic fungi Alternaria longipes resistance to dicarboximide fungicides （DCFs）, GeneFishing technology was conducted to analyse gene differential expression among A. longipes strains with different DCFs- resistant level, and the biological functions of the cloned AlCyP1 gene were analyzed by gene disruption. The results revealed that significant expression difference of a cyclophilin gene - AlCyP1 existed in different DCFs-resistant level strains. Using DNA walking method, the DNA sequence of AlCyP1 gene was cloned. Two translation initiation codons existed within the open reading frame of AlCyP1 gene, which coded two different length polypeptide products. The long and short polypeptide products contained 222 and 188 amino acids, respectively, in which the short one was initiated at codon 35 of the long polypeptide product. In addition, the sequence homology analysis of amino acids showed AlCyP1 sharing high homology （50.0%-61.3% ） with cyclophilins of other fungi. Finally, biological functions of the AlCyP1 gene were analyzed by gene disruption, and the results showed that AlCyP1 gene involved in A. longipes osmotic stress adaptation process dependent of its expression level change.

关 键 词：长柄链格孢 基因差异表达 亲环蛋白AlCyP1 渗透胁迫 二甲酰亚胺类杀菌剂 

分 类 号：Q939.95[生物学—微生物学]