摘要
采用逆转录聚合酶链式反应(RT-PCR)方法从耐玉米穗粒腐病自交系R15中分离得到病程相关蛋白1(pathogenesis-related protein 1,PR1)基因的开放阅读框,命名为ZmPR1,测序结果显示该序列长为528bp,编码175个氨基酸,其蛋白质分子质量为18.7ku.利用NCBI/Blastp和Genedoc软件进行同源性比对显示,ZmPR1基因编码蛋白与水稻、小麦、拟南芥等高等植物中的PR1蛋白相似性较高,且具有相同的富含半胱氨酸蛋白(cysteine-rich secretory protein,CAP)的保守结构域.将构建的重组载体pET32a(+)-ZmPR1在宿主菌Escherichia coli BL21中经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白,在不同的诱导时间、诱导温度和IPTG诱导浓度下对诱导条件进行优化.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明,ZmPR1基因的最佳诱导条件为IPTG终浓度0.6mmol爛L-1,诱导温度28℃,且诱导时间对表达量影响不大.免疫印迹(Western blot)检测证实有39ku的融合蛋白表达,表明ZmPR1基因在大肠杆菌中已成功表达,这为蛋白纯化及单克隆抗体的制备提供了一定的基础.
By reverse transcription-polymerase chain reaction（RT-PCR）,an open reading frame of pathogenesis-related protein 1（PR1） was isolated from inbred line R15 which was resistant to kernel rot,named ZmPR1.Molecular and bioinformatic analyses of ZmPR1 revealed that an open reading frame of 528 bp was predicted to encode a 175-amino acid protein with a deduced molecular mass of 18.7 ku.Homology analysis showed that the deduced amino acid sequence of PR1 protein in maize had a high similarity with other higher plants,such as Oryza sativa,Triticum aestivum,and Arabidopsis.PR1 proteins from different plants had the same conservative structure domain of cysteine-rich secretory protein（CAP）.The recombinant expressed plasmid pET32a（＋）-ZmPR1 was expressed in Escherichia coli BL21.The expression conditions were optimized by induction at different time,different temperature,and different IPTG concentrations.The results indicated that the expression products migrated at the size of 39 ku by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot analysis.The optimum expression condition was 0.6 mmol·L-1 IPTG at 28 ℃,but the induction time has little effect on the expression.The successful expression of ZmPR1 provides some basis for protein purification and preparation of the monoclonal antibody.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2012年第1期35-42,共8页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
国家自然科学基金资助项目(0900901)
农业部"高产转基因玉米新品种培育"资助项目(2008ZX08003-003)
四川省教育厅重点资助项目(07ZA063)
关键词
玉米穗粒腐病
病程相关蛋白
原核表达
表达分析
maize kernel rot
pathogenesis-related protein
prokaryotic expression
expression analysis
