三个甘蓝WRKY基因的克隆及其对非生物胁迫的表达

doi:10.13560/j.cnki.biotech.bull.1985.2023-0133

摘要/Abstract

摘要：

WRKY蛋白质是一类参与植物生长发育、生物和非生物胁迫以及其他生物过程的转录因子。研究甘蓝WRKY基因在逆境胁迫下的响应机制，为进一步研究BoWRKYs的抗逆功能奠定基础。以甘蓝（Brassica oleracea var. capitata L.）幼苗为试材，RT-PCR克隆BoWRKY40（BolC02g035760.2J）、BoWRKY46（BolC04g031460.2J）和BoWRKY70（BolC04g035730.2J）3个BoWRKYs；采用同源重组法构建pSuper1300: BoWRKYs -GFP载体，进行亚细胞定位；实时荧光定量PCR检测BoWRKYs在ABA、PEG8000和NaCl胁迫下的响应模式。结果表明，BoWRKY40、BoWRKY46和BoWRKY70的cDNA全长分别为873、831和864 bp，分别编码290、276和287个氨基酸，预测蛋白分子量为32.41、32.08和32.52 kD，理论等电点为6.77、6.18和5.62；多重比对分析发现3个BoWRKY蛋白结构域与拟南芥、番茄、玉米和棉花的WRKY结构域高度相似；亚细胞定位显示，3个BoWRKY蛋白主要定位于细胞核；荧光定量PCR分析显示，BoWRKYs在ABA、PEG8000和NaCl胁迫下受到不同程度的诱导。其中，BoWRKY40能积极响应3种胁迫，而BoWRKY46和BoWRKY70在ABA和盐胁迫下表达上调，在PEG8000处理后表达水平逐渐降低。甘蓝BoWRKY40、BoWRKY46和BoWRKY70表达与逆境胁迫响应关系密切。

关键词: 甘蓝, 基因克隆, 非生物胁迫, 亚细胞定位, 表达分析

Abstract:

WRKY proteins are a class of transcription factors involved in plant growth and development, biotic and abiotic stress, and other biological processes. To study the response mechanism of cabbage WRKY genes under stress of adversity may lay the foundation for further research on the stress tolerance function of BoWRKYs. BoWRKY40（BolC02g035760.2J）, BoWRKY46（BolC04g031460.2J）, and BoWRKY70（BolC04g035730.2J）from cabbage（Brassica oleracea var. capitata L.）seedlings were cloned by PCR approach. The pSuper1300: BoWRKYs-GFP expression vector was constructed by homologous recombination method for subcellular localization. Response patterns of BoWRKYs genes to abscisic acid（ABA）, PEG8000 and salt stress were analyzed by RT-qPCR. As results, the length of CDS regions of BoWRKY40, BoWRKY46 and BoWRKY70 were 873, 831 and 864 bp, which encoded proteins of 290, 276 and 287 amino acids, respectively. The predicted molecular weights of BoWRKY proteins were 32.41, 32.08 and 32.52 kD with theoretical isoelectric points（pI）of 6.77, 6.18 and 5.62. The conserved structural domain of the WRKY superfamily ‘WRKYGQK’ was present in the proteins, and multiple alignment analysis revealed that BoWRKYs had high conservation with WRKYs in Arabidopsis（Arabidopsis thaliana）, tomato（Solanum lycopersicum L.）, maize（Zea mays L.）, and cotton（Gossypium hirsutum L.）. Subcellular localization analysis showed that BoWRKYs were located in the nucleus. Fluorescence quantitative PCR results showed that WRKY family genes were induced in different degrees under ABA, PEG8000 and salt stress. Among them, BoWRKY40 responded positively to three kinds of stress, BoWRKY46 and BoWRKY70 were up-regulated under ABA and salt stress, and their expressions gradually decreased after PEG8000 treatment. These results indicated that the expressions of cabbage BoWRKY40, BoWRKY46, and BoWRKY70 were closely related to stress responses.

Key words: cabbage, gene cloning, abiotic stress, subcellular localization, expression analysis

杨旭妍, 赵爽, 马天意, 白玉, 王玉书. 三个甘蓝WRKY基因的克隆及其对非生物胁迫的表达[J]. 生物技术通报, 2023, 39(11): 261-269.

YANG Xu-yan, ZHAO Shuang, MA Tian-yi, BAI Yu, WANG Yu-shu. Cloning of Three Cabbage WRKY Genes and Their Expressions in Response to Abiotic Stress[J]. Biotechnology Bulletin, 2023, 39(11): 261-269.

图/表 8

表1 引物列表

Table 1 List of primers

应用Application	引物名称Primer name	引物序列Primer sequence（5'-3'）
基因克隆Gene cloning	BoWRKY40	F: ATGGACCAGTACCCATCGTCTTTG R: CTACTTGTCGGTTTGATTCTGTTGG
	BoWRKY46	F: ATGTTAATGGAAGAAAAGCTTGTG R: TTACATCCACGACAAATCTTGAG
	BoWRKY70	F: ATGGATATTGTTAGTAATAACAAAGC R: TCATTGTCGTTGAACCGGAAATC
实时荧光定量PCR Real-time PCR	qBoWRKY40	F: GGTGCTGCCAACAGAAGTAGGAG R: GGGCTTGTCACTTTCTTGGTTTCAG
	qBoWRKY46	F: AAGAGGTTGCTTGGCGGTGAAG R: AGAGTGTTTGGTGGTTGATGGAGTG
	qBoWRKY70	F: AACCCATCCCCTCCTATCTCTTCAC R: ATCCTCAAGTTTGCCGTCGTTGG
	BoActin2	F: CGTGACCTTACTGACTACC R: CTCCATCTCCTGCTCGTA
表达载体Expression vector	BoWRKY40	F: ctgcaggggcccggggtcgacATGGACCAGTACCCATCGTCTTTG R: gcccttgctcaccatggtaccCTACTTGTCGGTTTGATTCTGTTGG
	BoWRKY46	F: ctgcaggggcccggggtcgaccATGTTAATGGAAGAAAAGCTTGTG R: gcccttgctcaccatggtaccTTACATCCACGACAAATCTTGAG
	BoWRKY70	F:ctgcaggggcccggggtcgacATGGATATTGTTAGTAATAACAAAGC R: gcccttgctcaccatggtaccTCATTGTCGTTGAACCGGAAATC

图1 3个BoWRKYs基因cDNA的PCR扩增结果

Fig. 1 PCR amplification results of cDNAs from three BoWRKYs

图2 3个BoWRKYs顺式作用元件分析 ABRE：脱落酸响应；ARE：厌氧诱导；AuxRR-core：生长素响应；CAT-box：分生组织表达；CGTCA-motif：茉莉酸甲酯响应；G-box：光响应；MBS：参与干旱诱导的MYB结合位点； TCA-element：水杨酸响应； GARE-motif：赤霉素响应；LTR：低温响应；TC-rich repeats：防御和应激反应；W-box：WRKY结合位点

Fig. 2 Cis-elements of three BoWRKY genes ABRE: Abscisic acid responsiveness; ARE: anaerobic induction; AuxRR-core: auxin responsiveness; CAT-box: meristem expression; CGTCA-motif: MeJA-responsiveness; G-box: light responsiveness; MBS: MYB binding site involved in drought-inducibility; TCA-element: salicylic acid responsiveness; GARE-motif: gibberellin-responsive; LTR: low-temperature responsiveness; TC-rich repeats: defense and stress responsiveness; W-box: WRKY binding site

图3 3个BoWRKYs与其他物种WRKY保守结构域多序列比对 Bo：甘蓝；Bra：油菜；At：拟南芥；Gh：陆地棉。下同

Fig. 3 Multiple alignment of conserved domains of 3 BoWRKYs and WRKY from other plant species Bo: B. oleracea var. capitata; Bra: Brassica napus; At: Arabidopsis thaliana; Gh: Gossypium hirsutum. The same below

图4 BoWRKYs与其他物种WRKY蛋白系统的进化树分析 Sl：番茄；St：马铃薯

Fig. 4 A phylogenetic tree of BoWRKYs and WRKY proteins from other plant species Sl: Solanum lycopercicum; St: Solanum tuberosum

图5 构建pSuper1300: BoWRKYs-GFP植物表达载体

Fig. 5 Constructing pSuper1300: BoWRKYs -GFP expression vector in plants

图6 BoWRKYs的亚细胞定位

Fig. 6 Subcellular localization of BoWRKYs

图7 BoWRKYs在非生物胁迫下的表达分析图中误差线表示标准误差。小写字母表示不同处理时间基因表达量达到（P<0.05）显著水平

Fig. 7 Expression patterns of BoWRKYs gene under abiotic stresses The error line in the figure refers to the standard deviation. The lowercase letters indicate that the gene expression level reached（P<0.05）significant level at different treatment times

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