Cloning of Three Cabbage WRKY Genes and Their Expressions in Response to Abiotic Stress

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

Abstract

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

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.

Figures/Tables 8

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

Fig. 1 PCR amplification results of cDNAs from three BoWRKYs

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

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

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

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

Fig. 6 Subcellular localization of BoWRKYs

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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