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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2012, Vol. 38 Issue (1): 35-42    DOI: 10.3785/j.issn.1008-9209.2012.01.005
生物科学与技术     
玉米病程相关蛋白1基因的克隆与表达分析
王静１ , 刘丽１ , 张志明１ , 赵茂俊２ , 潘光堂１
１ .四川农业大学玉米研究所/教育部作物基因资源与遗传改良重点实验室/农业部西南玉米生物学与
遗传育种重点实验室,四川温江６１１１３０ ;２ .四川农业大学生命科学与理学院,四川雅安６２５０１４
Cloning and expression analysis of pathogenesis‐related protein 1 gene in maize
WANG Jing１ , LIU Li１ , ZHANG Zhi‐ming１ , ZHAO Mao‐jun２ , PAN Guang‐tang１
1 . M aize Research Institute / Key L aboratory o f Crop Genetic Resources and Imp rovement o f M inistry o f Education /
Key L aboratory o f Biology and Genetic Imp rovement o f M aize f or Southwest China , M inistry o f
A griculture , Sichuan A gricultural University , W enj iang , Sichuan 611130 , China ; 2 . College o f L i f e
and Basic Sciences , Sichuan A gricultural University , Y a′an , Sichuan 625014 , China
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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５２８ bp ,编码１７５ 个氨基酸,其蛋白质分子质量为１８ .７ ku .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.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３９ ku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.
Abstract: 　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 Oryz a sativ a , T riticum aestivum , and A rabidopsis . 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 .
出版日期: 2012-01-20
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王静
刘丽
张志明
赵茂俊
潘光堂

引用本文:

王静,刘丽,张志明,赵茂俊,潘光堂. 玉米病程相关蛋白1基因的克隆与表达分析[J]. 浙江大学学报(农业与生命科学版), 2012, 38(1): 35-42.

WANG Jing,LIU Li,ZHANG Zhiming,ZHAO Maojun,PAN Guangtang. Cloning and expression analysis of pathogenesis‐related protein 1 gene in maize. , 2012, 38(1): 35-42.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.01.005    或    http://www.zjujournals.com/agr/CN/Y2012/V38/I1/35

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