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.

参考文献 30

[1]	Laboissiere M C A,Sturley S L,Raines R T.The essential function of protein-disulfide isomerase is to unscramble non-native disulfide bonds[J].Journal of Biological Chemistry,1995,207(47):28006-28009.
[2]	Hunter B G,Beatty M K,Singletary G W,et al.Maize opaque endosperm mutations create extensive changes in patterns of gene expression[J].The Plant Cell,2002,14(10):2591-2612.
[3]	Meiri E,Levitan A,Guo F,et al.Characterization of three PDI-like genes in Physcomitrella patens and construction of knock-out mutants[J].Molecular Genetics and Genomics,2002,267(2):231-240.
[4]	Gruber C W,Cemažar M,Heras B,et al.Protein disulfide isomerase:the structure of oxidative folding[J].Trends in Biochemical Sciences,2006,31(8):455-464.
[5]	Wang L,Wang X,Wang C C.Protein disulfide-isomerase,a folding catalyst and a redox-regulated chaperone[J].Free Radical Biology and Medicine,2015,83:305-313.
[6]	Lu D P,Christopher D A.Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana[J].Molecular Genetics and Genomics,2008,280(3):199-210.
[7]	Narindrasorasak S,Yao P,Sarkar B.Protein disulfide isomerase,a multifunctional protein chaperone,shows copper-binding activity[J].Biochemical and Biophysical Research Communications,2003,311(2):405-414.
[8]	Hatahet F,Ruddock L W.Protein disulfide isomerase:a critical evaluation of its function in disulfide bond formation[J].Antioxidants & Redox Signaling,2009,11(11):2807-2850.
[9]	Klappa P,Stromer T,Zimmermann R,et al.A pancreas-specific glycosylated protein disulphide-isomerase binds to misfolded proteins and peptides with an interaction inhibited by oestrogens[J].European Journal of Biochemistry,1998,254(1):63-69.
[10]	Ferrari D M,Söling H D.The proteindisulphide-isomerase family:unravelling a string of folds[J].Biochemical Journal,1999,339:1-10.
[11]	Xu S L,Sankar S,Neamati N.Protein disulfide isomerase:a promising target for cancer therapy[J].Drug Discovery Today,2014,19(3):222-240.
[12]	Coe H,Michalak M.ERp57,a multifunctional endoplasmic reticulum resident oxidoreductase[J].The International Journal of Biochemistry & Cell Biology,2010,42(6):796-799.
[13]	刘颖慧,王秀堂,石云素,等.玉米蛋白质二硫键异构酶(PDI)基因的特征和表达[J].中国生物化学与分子生物学报,2009,25(3):229-234.Liu Y H,Wang X T,Shi Y S,et al.Expression and characterization of a protein disulfide isomerases in maize(Zea mays L.)[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(3):229-234.
[14]	Ciaffi M,Paolacci A R,D'aloisio E,et al.Cloning and characterization of wheat PDI(protein disulfide isomerase) homoeologous genes and promoter sequences[J].Gene,2006,366(2):209-218.
[15]	王秀堂,黄亚群,李会勇,等.玉米PDI基因cDNA的克隆及生物信息学分析[J].河北农业大学学报,2008,31(1):16-19.Wang X T,Huang Y Q,Li H Y,et al.cDNA cloning and bioinformatic analysis of protein disulfide isomerase gene of maize(Zea mays L.)[J].Journal of Agricultural University of Hebei,2008,31(1):16-19.
[16]	D'aloisio E,Paolacci A R,Dhanapal A P,et al.The protein disulfide isomerase gene family in bread wheat(Triticum aestivum L.)[J].BMC Plant Biology,2010,10:101.
[17]	Wu H M,Dorse S,Bhave M.In silico identification and analysis of the protein disulphide isomerases in wheat and rice[J].Biologia,2012,67(1):48-60.
[18]	Han X H,Wang Y H,Liu X,et al.The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice[J].Journal of Experimental Botany,2012,63(1):121-130.
[19]	Yuen C Y,Matsumoto K O,Christopher D A.Variation in the subcellular localization and protein folding activity among Arabidopsis thaliana homologs of protein disulfide isomerase[J].Biomolecules,2013,3(4):848-869.
[20]	张政荣.拟南芥蛋白质二硫键异构酶(AtPDIL1)酶活性及其抗逆功能的研究[D].泰安:山东农业大学,2017.Zhang Z R.Enzyme activity and resistance function analysis of protein disulfide isomerase(At PDIL1) from Arabidopsis[D].Taian:Shandong Agricultural University,2017.
[21]	Satoh-Cruz M,Crofts A J,Takemoto-Kuno Y,et al.Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm[J].Plant and Cell Physiology,2010,51(9):1581-1593.
[22]	Takemoto Y,Coughlan S J,Okita T W,et al.The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase[J].Plant Physiology,2002,128(4):1212-1222.
[23]	Joye I J,Lagrain B,Delcour J A.Endogenous redox agents and enzymes that affect protein network formation during breadmaking-a review[J].Journal of Cereal Science,2009,50(1):1-10.
[24]	Huang D J,Chen H J,Lin Y H.Isolation and expression of protein disulfide isomerase cDNA from sweet potato(Ipomoea batatas L. Lam ‘Tainong 57’) storage roots[J].Plant Science,2005,169(4):776-784.
[25]	Xu Z J,Ueda K J,Masuda K,et al.Molecular characterization of a novel proteindisulfide isomerase in carrot[J].Gene,2002,284(1-2):225-231.
[26]	Roustan V,Roustan P J,Weidinger M,et al.Microscopic and proteomic analysis of dissected developing barley endosperm layers reveals the starchy endosperm as prominent storage tissue for ER-Derived hordeins alongside the accumulation of barley protein disulfide Isomerase(HvPDIL1-1)[J].Frontiers in Plant Science,2018,9:1248.
[27]	Zhang Z R,Liu X,Li R,et al.Identification and functional analysis of a protein disulfide isomerase(AtPDI1) in Arabidopsis thaliana[J].Frontiers in Plant Science,2018,9:913.
[28]	Lan X G,Yang J,Abhinandan K,et al.Flavonoids and ROS Play opposing roles in mediating pollination in ornamental kale(Brassica oleracea var.acephala)[J].Molecular Plant,2017,10(10):1361-1364.
[29]	Lan X G,Yang J,Cao M M,et al.Isolation and characterization of a J domain protein that interacts with ARC1 from ornamental kale(Brassica oleracea var.acephala)[J].Plant Cell Reports,2015,34(5):817-829.
[30]	Li X,Abhinandan K,Zhang T,et al.Temporal regulation of two cytosolic phosphoglucomutases during stigmadevelopment in ornamental kale(Brassica oleracea var.acephala)[J].Plant Signaling & Behavior,2018,13(5):e1467698.

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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