羽衣甘蓝类蛋白质二硫键异构酶PDIL1-2的基因克隆及蛋白表达分析  

Gene Cloning and Expression Analysis of PDIL1-2 of Ornamental Kale(Brassica oleracea var.acephala)

作　　者：李阳[1] 施亚坤 高士科 李晓屿 蓝兴国[1] LI Yang;SHI Ya-Kun;GAO Shi-Ke;Li Xiao-Yu;LAN Xing-Guo(College of Life Science,Northeast Forestry University,Harbin 150040)

机构地区：[1]东北林业大学生命科学学院,哈尔滨150040

出　　处：《植物研究》2020年第2期293-300,共8页Bulletin of Botanical Research

基　　金：黑龙江省大学生创新创业训练计划项目(201810225338);中央高校基本科研业务费专项基金项目(2572018BD01);国家自然科学基金面上项目(31870300)。

摘　　要：以羽衣甘蓝(Brassica oleracea var.acephala)自交不亲和系(S13-bS13-b)为试材,利用RT-PCR和RACE的方法从柱头中分离类蛋白质二硫键异构酶BoPDIL1-2基因。将BoPDIL1-2编码区的序列构建到原核表达载体pET-14b上,并转化到大肠杆菌中进行原核表达与纯化;用纯化后的蛋白免疫小鼠,制备BoPDIL1-2多克隆抗体;通过免疫印迹法检测BoPDIL1-2在不同组织及不同发育阶段柱头的表达情况。氨基酸序列比对分析结果显示,羽衣甘蓝BoPDIL1-2与油菜BnPDIL1-2、拟南芥AtPDIL1-2的一致性分别是97.3%和85.5%。SDS-PAGE结果显示,在分子量58 kD处有BoPDIL1-2蛋白特异性地诱导表达。免疫印迹结果显示BoPDIL1-2在羽衣甘蓝柱头中特异表达,而且在柱头发育早期表达量较低,在开花期柱头表达量较高。The BoPDIL1-2 gene was isolated from the stigma of ornamental kale(Brassica oleracea var.acephala)S13-bS13-b homozygotes by RT-PCR and RACE.The BoPDIL1-2 coding region sequence was inserted into the prokaryotic expression vector pET-14b and transformed into the E.coli cell for purification.We prepared the polyclonal antibodies against recombinant BoPDIL1-2 through immune mouse.The expression level of BoPDIL1-2 in the various tissue anddifferent developmental stigmas were detected by Western blot.The deduced amino acid sequence of BoPDIL1-2 shared 97.3%and 85.5%identity with Brassica napus BnPDIL1-2 and Arabidopsis thaliana AtPDIL1-2,respectively.SDS-PAGE results showed that the recombinant BoPDIL1-2 fusion protein about 58 kDa was induced by IPTG.Western blot results revealed BoPDIL1-2 wasspecifically expressed inthe stigma,the expression of BoPDIL1-2 was lower in the early stage of stigma,while it was higher in the stigma at theflowering stage.

关 键 词：羽衣甘蓝 类蛋白质二硫键异构酶 原核表达 多克隆抗体 表达分析 

分 类 号：S635[农业科学—蔬菜学]