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

doi:10.7525/j.issn.1673-5102.2020.02.017

植物研究 ›› 2020, Vol. 40 ›› Issue (2): 293-300.doi: 10.7525/j.issn.1673-5102.2020.02.017

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

李阳, 施亚坤, 高士科, 李晓屿, 蓝兴国

东北林业大学生命科学学院, 哈尔滨 150040

收稿日期:2019-07-31 出版日期:2020-03-05 发布日期:2020-03-06
通讯作者: 蓝兴国,E-mail:lanxingguo@126.com E-mail:lanxingguo@126.com
作者简介:李阳(1999-),女,本科生,主要从事植物发育方向的研究。
基金资助:
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）

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

LI Yang, SHI Ya-Kun, GAO Shi-Ke, Li Xiao-Yu, LAN Xing-Guo

College of Life Science, Northeast Forestry University, Harbin 150040

Received:2019-07-31 Online:2020-03-05 Published:2020-03-06
Supported by:
Heilongjiang Student's Platform for Innovation and Entrepreneurs(201810225338);Fundamental Research Funds for the Central Universities(2572018BD01);National Natural Science Foundation Project(31870300)

摘要/Abstract

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。

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

Abstract: The BoPDIL1-2 gene was isolated from the stigma of ornamental kale(Brassica oleracea var. acephala) S_13-bS_13-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.

Key words: Brassica oleracea var. acephala, PDIL1-2, prokaryotic expression, polyclonal antibody, expression analysis

中图分类号:

S635

李阳, 施亚坤, 高士科, 李晓屿, 蓝兴国. 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[J]. 植物研究, 2020, 40(2): 293-300.

LI Yang, SHI Ya-Kun, GAO Shi-Ke, Li Xiao-Yu, LAN Xing-Guo. Gene Cloning and Expression Analysis of PDIL1-2 of Ornamental Kale(Brassica oleracea var. acephala)[J]. Bulletin of Botanical Research, 2020, 40(2): 293-300.

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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)

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