西兰花灰霉病响应基因BoWRKY15的克隆与功能鉴定

doi:10.3969/j.issn.1004-1524.20240941

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (8): 1723-1732.DOI: 10.3969/j.issn.1004-1524.20240941

西兰花灰霉病响应基因BoWRKY15的克隆与功能鉴定

蒋明¹(), 张胜², 陈孝赏³, 张慧娟¹^,^*()

1.台州学院生命科学学院, 浙江台州 318000
2.台州市农业技术推广中心,浙江台州 318000
3.台州市农业科学研究院蔬菜研究所,浙江台州 318000

收稿日期:2024-11-05 出版日期:2025-08-25 发布日期:2025-09-03
作者简介:蒋明(1973—),男,浙江嵊州人,博士,教授,研究方向为植物逆境生物学与分子调控。E-mail:jiangming1973@139.com
通讯作者: *张慧娟,E-mail:zhanghj82@126.com
基金资助:
台州市科技计划(22nya01);浙江省自然科学基金(LY19C150004)

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

摘要/Abstract

摘要： 灰葡萄孢菌(Botrytis cinerea)引起的灰霉病是西兰花(Brassica oleracea var. italica)生产中的重要病害之一,每年造成的经济损失巨大。WRKY转录因子是植物特有的一类调控蛋白,在逆境防御中起重要作用。本研究以西兰花为材料,在克隆BoWRKY15基因的基础上,利用生物信息学手段明确BoWRKY15的序列特征,采用qRT-PCR技术检测其在灰葡萄孢菌侵染下的表达模式,并利用过表达技术明确转基因西兰花的抗病性变化,从而明确该基因在抗病响应中的功能。结果表明,BoWRKY15的基因组全长为1 172 bp,有2个内含子,分别为90 bp和86 bp;编码区全长为996 bp,编码331个氨基酸,包含1个由61个氨基酸组成的WRKY结构域,其锌指结构类型为C2H2。多重序列比对发现,BoWRKY15及其同源序列的WRKY结构域十分保守,仅个别氨基酸残基存在差异。系统发育分析显示,BoWRKY15与来自芸薹属植物的同源序列聚于一组。亚细胞定位结果表明,BoWRKY15在细胞核中表达,与在线预测结果一致。qRT-PCR结果表明,BoWRKY15的表达受灰葡萄孢菌诱导,在接种48 h时的表达量最大,为对照组的2.64倍。BoWRKY15过表达导致西兰花对灰葡萄孢菌的抗性显著降低,同时病程相关蛋白基因BoiPR1的表达量升高。BoWRKY15在灰霉病抗性响应中起负调控作用,该基因的克隆与表达分析为后续开展西兰花抗病机理研究和分子育种奠定了基础。

关键词: 西兰花, WRKY转录因子, 过表达, 灰霉病, 灰葡萄孢菌

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

中图分类号:

S436.35

蒋明, 张胜, 陈孝赏, 张慧娟. 西兰花灰霉病响应基因BoWRKY15的克隆与功能鉴定[J]. 浙江农业学报, 2025, 37(8): 1723-1732.

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.

图/表 9

图1 BoWRKY15基因及其编码的氨基酸序列下划线序列为HARF结构域;方框部分为核定位信号序列;高亮区域为WRKY结构域。

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.

图2 BoWRKY15及其同源蛋白的WRKY结构域

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

图3 基于BoWRKY15及其同源蛋白构建的系统发育树

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

图4 BoWRKY15基因在灰葡萄孢菌诱导下的表达量柱上无相同小写字母表示差异显著(p<0.05)。下同。

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.

图5 BoWRKY15的亚细胞定位

Fig.5 The subcellular localization of BoWRKY15

图6 BoWRKY15过量表达植株的PCR鉴定 M,DNA marker;P,阳性质粒;WT,野生型;1~16,转基因株系。

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

图7 BoWRKY15基因在不同西兰花株系中的相对表达量 WT,野生型;WK02-10-09和WK12-11-24,BoWRKY15基因过表达株系。下同。

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.

图8 不同株系西兰花叶片上的灰霉病病斑直径

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

图9 BoiPDF1.2和BoiPR1基因在不同西兰花株系中的表达量

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

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西兰花灰霉病响应基因BoWRKY15的克隆与功能鉴定

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

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