ChiC基因调控番茄灰霉病抗性的机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1242

摘要/Abstract

摘要：

目的灰葡萄孢（Botrytis cinerea）引起的灰霉病严重威胁番茄等设施园艺作物的生产与采后安全，且目前植物自身可利用的抗病基因资源相对匮乏。以几丁质酶为切入点，解析几丁质酶合成基因ChiC调控番茄灰霉病抗性机制，为探究几丁质酶在番茄响应灰霉病过程中的功能奠定基础。方法以番茄为材料，利用CRISPR/Cas9技术构建ChiC基因突变体，并通过农杆菌介导法获得ChiC过表达株系。对上述遗传材料进行灰霉菌接种实验，对不同遗传背景番茄材料的果实和叶片进行抗病表型分析与病斑面积统计，以验证ChiC基因在抗病过程中的功能。同时，通过RT-qPCR分析灰霉菌侵染后不同番茄遗传材料中防御相关基因的表达变化。结果序列和结构分析发现，番茄ChiC基因编码1个含376个氨基酸残基的几丁质酶蛋白，该蛋白属于GH18亚家族。定量结果显示，灰霉菌侵染番茄果实能够显著诱导ChiC基因的表达。灰霉菌侵染试验结果表明，与野生型相比，chic突变体植株的果实和叶片对灰霉菌更加感病，而ChiC过表达植株则表现出显著增强的抗病性。进一步的基因表达分析发现，在灰霉菌侵染后，ChiC过表达植株中ERF.C3、PR-STH2d和PR-STH2c等防御基因的表达显著上调。结论番茄ChiC基因可正向调控番茄对灰霉病的抗性，其作用机制可能包括直接降解真菌细胞壁及通过诱导防御基因表达增强植物免疫反应。

关键词: 番茄, 灰霉病, 灰葡萄孢, 几丁质酶, ChiC, CRISPR/Cas9, 抗病性, 防御基因

Abstract:

Objective Gray mold caused by Botrytis cinerea poses a serious threat to the production and postharvest safety of tomato and other protected horticultural crops, while exploitable disease-resistance gene resources in plants remain limited. In this study, focusing on chitinase, we aim to elucidate the regulatory mechanism of the chitinase synthesis gene ChiC in tomato resistance to gray mold, thereby providing a basis for understanding the role of chitinases in tomato responses to B. cinerea. Method Using tomato as the experimental material, the ChiC knockout mutants were generated by CRISPR/Cas9 technology, and ChiC overexpression lines were obtained via Agrobacterium-mediated transformation. B. cinerea inoculation assays were performed on these genetic materials, and disease phenotypes and lesion areas on fruits and leaves from different genetic backgrounds were analyzed to verify the role of ChiC in disease resistance. Meanwhile, RT-qPCR analysis was performed to examine the expression variations of defense genes in different tomato genotypes after B. cinerea infection. Result Sequence and structural analyses revealed that the tomato ChiC gene encodes a chitinase consisting of 376 amino acids, which belongs to the GH18 subfamily. Quantitative analyses showed that infection of tomato fruits by B. cinerea significantly induces ChiC expression. Disease assays demonstrated that, compared with wild-type plants, the chic mutant plants have increased susceptibility of both fruits and leaves to B. cinerea, whereas ChiC overexpression plants demonstrate markedly enhanced disease resistance. Further gene expression analysis revealed that in ChiC overexpression plants, the expression of defense-related genes such as ERF.C3, PR-STH2d, and PR-STH2c were significantly upregulated following B. cinerea infection. Conclusion The tomato ChiC gene positively regulates tomato resistance to B. cinerea, and its mode of action may involve directly degrading fungal cell walls and enhancing plant immune responses through the induction of defense gene expression.

Key words: tomato, gray mold, Botrytis cinerea, chitinase, ChiC, CRISPR/Cas9, disease resistance, defense-related gene

李亚妮, 韩鸿宇, 耿梦爽, 米若兰, 王韦琪, 于文静, 孟宪文, 李传友. ChiC基因调控番茄灰霉病抗性的机制研究[J]. 生物技术通报, 2026, 42(3): 255-262.

LI Ya-ni, HAN Hong-yu, GENG Meng-shuang, MI Ruo-lan, Wang Wei-qi, Yu Wen-jing, MENG Xian-wen, LI Chuan-you. Mechanistic Study on ChiC-mediated Regulation Mechanism of Tomato Resistance to Botrytis cinerea[J]. Biotechnology Bulletin, 2026, 42(3): 255-262.

图/表 5

表1 实验所用定量引物

Table 1 Primers for RT-qPCR used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）
ACTIN2-qRT-F	TTGCTGACCGTATGAGCAAG
ACTIN2-qRT-R	GGACAATGGATGGACCAGAC
ChiC-qRT-F	TGGACTTGATCTTGATTGGGAA
ChiC-qRT-R	CCGGATAGTTCAAACCATTGAC
ERF.C3-qRT-F	GAATTCGTCGTCTAGCTATGGA
ERF.C3-qRT-R	GAACCTCTCATAGCAAATGCAG
PR-STH2c-qRT-F	AATCCTTCTGTCTACGCTTGAA
PR-STH2c-qRT-R	TTAACATCACAACACGTTCACG
PR-STH2d-qRT-F	AATAAGAATCAGGTCCACACGT
PR-STH2d-qRT-R	CACACAACTCAACAAAAAGCAC

图1 番茄ChiC结构及基因表达模式A：番茄与拟南芥几丁质酶基因的系统发育树；B：ChiC蛋白结构域；C：ChiC三维结构；D：ChiC基因在番茄不同组织中的表达（IMG：未熟绿期；Br：破色期；Br7：破色期后第7天）；E：灰霉菌侵染果实前后的表达模式；F：灰霉菌侵染叶片前后的表达模式；柱状图的数值表示为平均值±标准误（n=3），不同小写字母表示在P<0.05水平上差异显著，下同

Fig. 1 Structure of tomato ChiC and its expression patternsA: Phylogenetic tree of chitinase genes from tomato and Arabidopsis. B: ChiC protein domains. C: Three-dimensional structure of ChiC. D: Relative expression of the ChiC gene in different tissues (IMG: immature green; Br: breaker; Br7: 7 d after breaker stage). E: Expression patterns of ChiC before and after B. cinerea infection in the fruits. F: Expression patterns of ChiC before and after B. cinerea infection in leaves. The values in the bar charts are presented as mean ± standard error (n=3). Different lowercase letters indicate significant differences at P<0.05 level. The same below

图2 ChiC遗传材料的创制及几丁质酶活性检测A：基因编辑靶点位置及chic-1和chic-2株系基因突变类型；B：chic-1和chic-2株系ChiC蛋白结构域；C：ChiC基因在过表达植株中的相对表达量；D：转基因材料中几丁质酶活性检测

Fig. 2 Generation of ChiC genetic materials and assays of chitinase activityA: Positions of the gene-editing targets and mutation types in the chic-1 and chic-2 lines. B: ChiC protein domain structure in the chic-1 and chic-2 lines. C: Relative expression of ChiC in overexpression lines. D: Detection of chitinase activity in transgenic materials

图3 ChiC正向调控番茄对灰霉病的抗性A：灰霉菌侵染破色期果实3 d后的发病症状及病斑面积统计；B：灰霉菌侵染红熟期果实3 d后的发病症状及病斑面积统计；C：灰霉菌侵染叶片3 d后的发病症状及病斑面积统计。Bar=1 cm

Fig. 3 ChiC positively regulates the resistance to B. cinerea in tomatoA: Disease symptoms and quantification of lesion areas in breaker-stage fruits at 3 d after B. cinerea infection. B: Disease symptoms and quantification of lesion areas in red-ripe fruits at 3 d after B. cinerea infection. C: Disease symptoms and quantification of lesion areas in leaves at 3 d after B. cinerea infection. Bar=1 cm

图4 ChiC灰霉菌侵染过程中调控防御基因的表达

Fig. 4 ChiC modulates the expressions of defense genes in response to B. cinerea infection

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