马铃薯软腐病菌Erwinia carotovora subsp.carotovora 714种胞外酶基因的克隆和原核表达分析  

Cloning and analyzing of prokaryotic expression of four exoenzyme genes from potato soft-rot pathogen Erwinia carotovora subsp.carotovora 71

作　　者：李洋[1,2] 张俊莲[1,2] 白江平[1,2] 徐振峰[2] 王蒂[1,2] 

机构地区：[1]甘肃省作物遗传改良与种质创新重点实验室-甘肃省干旱生境作物学重点实验室,甘肃兰州730070 [2]甘肃农业大学农学院,甘肃兰州730070

出　　处：《草业科学》2014年第4期561-574,共14页Pratacultural Science

基　　金：国家科技支撑计划(2012BAD06B03);甘肃省重大专项项目(1102NKDA025)

摘　　要：以马铃薯软腐病菌Erwinia carotovora subsp.carotovora71(Ecc71)为材料,利用同源克隆技术克隆软腐病果胶酸盐裂解酶(pel)、多聚半乳糖醛酸酶(peh)、纤维素酶(cel)和蛋白酶(prt)基因,对其进行生物信息学分析,并将其克隆到原核表达载体pET28a中,将重组载体pET28a-pel、pET28a-peh、pET28a-cel和pET28a-prt转化至大肠杆菌E.coli BL21(DE3),经IPTG诱导后,SDS-PAGE电泳鉴定和酶活测定,得到了与理论推算的pel、peh、cel和prt基因表达产物分子质量相符的特异条带,并且其酶活性分别为1.672U·mL-1·h-1和0.024、112.7、16.95U·mL-1·min-1。这些结果为今后探究这4种酶之间的相互作用及马铃薯抗病性研究奠定了基础。In the present study, the virulence genes pel, peh, eel and prt of Erwinia carotovora subsp. carotovora 71 （Ecc71） which was the main pathogens of potato soft rot were cloned by homologous cloning techniques. The sequences and predicted gene products were analyzed with bioinformatics. These four genes were cloned into the prokaryotic expression vector pET28a. The successful recombinant vectors pET28a-pel, pET28a-peh, pET28a-cel and pET28a prt were transformed into E. colt BI.21 （DE3） respectively. The expression products of pel and peh genes were analyzed by SDS-PAGE were also analyzed. The specific proteins with same molecular weights wit deduced genes were observed and the enzyme activity was l. 672 U ·mLi · t 1, 0. 02 U · mL 1 . rain , respectively. The results provided foundation for exloring th these four enzymes and the disease-resistance of the potato soft-rot. and enzyme activities proteins from studied 4, 112. 7 and 16. 95 e interaction between

关 键 词：马铃薯 软腐病 致病因子 克隆 原核表达 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]