Cloning and functional verification of the gray mold disease responsive gene BoWRKY15 in broccoli(Brassica oleracea var. italica)

doi:10.3969/j.issn.1004-1524.20240941

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (8): 1723-1732.DOI: 10.3969/j.issn.1004-1524.20240941

• Plant Protection • Previous Articles Next Articles

Cloning and functional verification of the gray mold disease responsive gene BoWRKY15 in broccoli(Brassica oleracea var. italica)

JIANG Ming¹(), ZHANG Sheng², CHEN Xiaoshang³, ZHANG Huijuan¹^,^*()

1. College of Life Sciences, Taizhou University, Taizhou 318000, Zhejiang, China
2. Taizhou Agricultural Technology Extension and Service Center, Taizhou 318000, Zhejiang, China
3. Vegetable Research Institute, Taizhou Academy of Agricultural Sciences, Taizhou 318000, Zhejiang, China

Received:2024-11-05 Online:2025-08-25 Published:2025-09-03
Contact: ZHANG Huijuan

Abstract

Abstract:

Gray mold disease caused by Botrytis cinerea is one of the major diseases affecting broccoli production, resulting in significant economic losses annually. WRKY transcription factor is a group of regulatory proteins found only in plants, playing an important role in stress defense. In this study, using broccoli as the experimental material, BoWRKY15 gene was isolated, and sequence characteristics were determined by bioinformatics methods; qRT-PCR was employed to detect its expression pattern under B. cinerea infection, and disease resistance ability was investigated by over-expression of BoWRKY15 in broccoli, thereby clarifying the function of this gene in the disease resistance response. Results revealed that the full length of BoWRKY15 gene was 1 172 bp with two introns measuring 90 bp and 86 bp, respectively, while the coding region was 996 bp and encoded 331 amino acids, including one WRKY structure composed of 61 amino acids, and a zinc finger of C2H2 type. Multiple sequence alignment results indicated that the WRKY domains of BoWRKY15 and its homologous sequences were highly conserved, with only a few differences in amino acid residues. Phylogenetic analysis indicated that BoWRKY15 clustered with homologous sequences from the Brassica genus. The subcellular localization results indicated that BoWRKY15 was expressed in the nucleus, consistent with the online prediction. Gene expression analysis demonstrated that BoWRKY15 was induced by B. cinerea inoculation, with a maximum expression level observed at 48 h post-inoculation, reaching 2.64-fold as compared with control plants. Furthermore, over expression of BoWRKY15 led to significantly decreased resistance to gray mold infection, concomitant with increased BoiPR1 expression level. BoWRKY15 acts as a negative regulator in the defense response to B. cinerea, and the cloning and expression analysis of BoWRKY15 lays a foundation for further investigation into the disease resistance mechanisms and molecular breeding of broccoli in the future.

Key words: Brassica oleracea var. italica, WRKY transcription factor, overexpression, gray mold disease, Botrytis cinerea

CLC Number:

S436.35

JIANG Ming, ZHANG Sheng, CHEN Xiaoshang, ZHANG Huijuan. Cloning and functional verification of the gray mold disease responsive gene BoWRKY15 in broccoli(Brassica oleracea var. italica)[J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1723-1732.

Figures/Tables 9

Fig.1 The BoWRKY15 gene and its encoded amino acid sequence The underlined sequence indicates the HARF domain; The boxed residues is the nuclear localization signal sequence; The highlighted region indicates the WRKY domain.

Fig.2 The WRKY domains of BoWRKY15 and its homologous proteins

Fig.3 The phylogenetic tree constructed based on BoWRKY15 and its homologous proteins

Fig.4 Expression of BoWRKY15 gene induced by Botrytis cinerea The bars marked without the same lowercase letter indicated significant differences (p<0.05). The same as below.

Fig.5 The subcellular localization of BoWRKY15

Fig.6 PCR identification of BoWRKY15 over-expression plants M, DNA marker; P, Positive plasmid; WT, Wild type; 1-16, Transgenic lines.

Fig.7 Relative expression level of BoWRKY15 gene in different broccoli lines WT, Wild type; WK02-10-09 and WK12-11-24, BoWRKY15 gene over-expression lines. The same as below.

Fig.8 Lesion diameter of gray mold disease on broccoli leaves from different lines

Fig.9 Expression levels of BoiPDF1.2 and BoiPR1 genes in different broccoli lines

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Cloning and functional verification of the gray mold disease responsive gene BoWRKY15 in broccoli(Brassica oleracea var. italica)

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