NtPLA2A介导激素信号通路增强烟草青枯病抗性的机制

doi:10.13560/j.cnki.biotech.bull.1985.2025-1168

摘要/Abstract

摘要：

目的克隆烟草NtPLA2A基因，探究其介导激素信号通路增强烟草青枯病抗性的分子机制。方法克隆了烟草NtPLA2A基因，对其进行生物信息学分析和青枯菌侵染下的表达分析，并探究NtPLA2A在本氏烟抵抗青枯菌入侵中的作用。结果结果表明，NtPLA2A基因编码区长1 284 bp，编码428个氨基酸。蛋白分子量为46.57 kD，理论等电点为8.74，为稳定亲水蛋白。生物信息学分析显示，NtPLA2A蛋白具有典型的Patatin-like phospholipase结构域，且与其他物种的PLA蛋白具有较高的氨基酸序列同源性。启动子分析表明，NtPLA2A基因启动子区域包含低温、光及激素等环境因子的作用元件。青枯菌侵染后，NtPLA2A基因在不同抗/感青枯病烟草材料中均受到不同程度的上调表达，且水杨酸、茉莉酸和乙烯处理也能诱导其上调表达。VIGS结果显示，沉默NtPLA2A基因降低了本氏烟草对青枯菌的抗性。结论明确了烟草NtPLA2A基因序列、结构及表达特征，其受青枯菌与SA/JA/ET诱导，沉默该基因后本氏烟抗青枯病能力显著下降。

关键词: 烟草, NtPLA2A基因, 青枯菌, 基因克隆, 病毒诱导的基因沉默

Abstract:

Objective To clone the NtPLA2A gene from tobacco (Nicotiana tabacum) and explore the molecular mechanism by which it mediates hormone signaling pathways to enhance tobacco resistance to bacterial wilt. Method This study cloned the NtPLA2A gene from tobacco, conducted bioinformatic analysis and expression profiling under Ralstonia solanacearum infection, and investigated the role of NtPLA2A in disease resistance using virus-induced gene silencing (VIGS) in N. benthamiana. Result The NtPLA2A coding sequence is 1 284 bp, encoding 428 amino acids. The protein has a molecular weight of 46.57 kD and a theoretical isoelectric point of 8.74, and is a stable hydrophilic protein. Bioinformatic analysis revealed that NtPLA2A contains a typical Patatin-like phospholipase domain and shares high amino acid sequence similarity with PLA proteins from other species. Promoter analysis indicated that the promoter region of NtPLA2A contains environmental factors such as light, low temperature, and hormones. Following R. solanacearum infection, NtPLA2A was significantly upregulated in both resistant and susceptible tobacco materials to varying degrees. Additionally, treatments with salicylic acid, jasmonic acid, and ethylene also induced its expression. VIGS results demonstrated that silencing NtPLA2A compromised resistance to R. solanacearum in N. benthamiana. Conclusion This study clarified the sequence, structure, and expression characteristics of tobacco NtPLA2A gene, which is induced by R. solanacearum and SA/JA/ET; its silencing significantly impairs N. benthamiana resistance to bacterial wilt.

Key words: tobacco, NtPLA2A gene, Ralstonia solanacearum, gene cloning, virus-induced gene silencing (VIGS)

张仲文, 王卫民, 肖志亮, 史久长, 郝贤伟, 王威, 谢鹏岗, 许佳丹, 赵炯平, 杨爱国. NtPLA2A介导激素信号通路增强烟草青枯病抗性的机制[J]. 生物技术通报, 2026, 42(8): 207-216.

ZHANG Zhong-wen, WANG Wei-min, XIAO Zhi-liang, SHI Jiu-chang, HAO Xian-wei, WANG Wei, XIE Peng-gang, XU Jia-dan, ZHAO Jiong-ping, YANG Ai-guo. Mechanism of NtPLA2A-mediated Hormone Signaling Pathway in Enhancing Tobacco Resistance to Bacterial Wilt[J]. Biotechnology Bulletin, 2026, 42(8): 207-216.

图/表 12

参考文献 34

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网站（网址） Website （URL）	分析用途 Purpose of analysis
ProtParam （https：//web.expasy.org/protparam）	预测蛋白的理化性质
ProtScale（https：//web.expasy.org/protscale）	预测蛋白的亲疏水性
SOPMA（https：//npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl page=npsa_sopm.html）	预测蛋白的二级结构
SWISS-MODEL（https：//swissmodel. expasy.org）	预测蛋白的三级结构
TMHMM-2.0（http：//www.csbio.sjtu.edu.cn/bioinf/MHMM/）	预测蛋白的跨膜结构域
Plant-mPLoc（http：//www.csbio.sjtu.edu.cn/bioinf/plantmulti）	预测蛋白的亚细胞定位
PlantCARE（http：//bioinformatics.psb.ugent.be/webtools/plantcare/html/）	预测基因启动子中的顺式作用元件

网站（网址） Website （URL）	分析用途 Purpose of analysis
ProtParam （https：//web.expasy.org/protparam）	预测蛋白的理化性质
ProtScale（https：//web.expasy.org/protscale）	预测蛋白的亲疏水性
SOPMA（https：//npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl page=npsa_sopm.html）	预测蛋白的二级结构
SWISS-MODEL（https：//swissmodel. expasy.org）	预测蛋白的三级结构
TMHMM-2.0（http：//www.csbio.sjtu.edu.cn/bioinf/MHMM/）	预测蛋白的跨膜结构域
Plant-mPLoc（http：//www.csbio.sjtu.edu.cn/bioinf/plantmulti）	预测蛋白的亚细胞定位
PlantCARE（http：//bioinformatics.psb.ugent.be/webtools/plantcare/html/）	预测基因启动子中的顺式作用元件

基因 Gene	沉默序列 Silencing sequence （5'-3'）
NtPLA2A	GGTAGAACTTTTGTAGCTGCACTAACTTTATTAGTGACTCTTCATGTTCTACTACCTGTTATGGTTTCTGCTGCTACAAAAGGAAAGATAGTAACAGTTTTGAGCATAGATGGAGGTGGCATCAGAGGCATTATTCCTGGCACCCTTCTTGCTTTCCTTGAATCCAAGCTTCAGGACATAGATGGACCGAATGCAAGAATTGCAGACTATTTTGATGTTGTAGCTGGAACGAGCACAGGTGGACTAATAAGCACCATGCTCACAGCTCCAAACAAGGATAATCGCCCTTTATATGCA
NtPDS	ATGCTAACTAGCTGGAGACTCTTTCCTGCTTCGTTCACCAGGCCTAAGTACTGCCCAAGTGGATTTTATTGCAGGGCTGAGGATCAACTGAGTAGTATAAATGAACGTCAAAAGAAGAAGAAAGTGCTGATAGTAGGTTCAGGCTGGGCTGGCCTTGGAGCTGCTCACCATCTCTGCAAACAGGGCTTTGAGGTCGTTGTTCTTGAAGGTGGCTATGAATTTGGACCCAAAAATCAATCCCTAAGCCCTGACGATGTGGCTATTCGCGGTTTCTGGTATCCCTATCGAAATATATTTGAT

基因 Gene	沉默序列 Silencing sequence （5'-3'）
NtPLA2A	GGTAGAACTTTTGTAGCTGCACTAACTTTATTAGTGACTCTTCATGTTCTACTACCTGTTATGGTTTCTGCTGCTACAAAAGGAAAGATAGTAACAGTTTTGAGCATAGATGGAGGTGGCATCAGAGGCATTATTCCTGGCACCCTTCTTGCTTTCCTTGAATCCAAGCTTCAGGACATAGATGGACCGAATGCAAGAATTGCAGACTATTTTGATGTTGTAGCTGGAACGAGCACAGGTGGACTAATAAGCACCATGCTCACAGCTCCAAACAAGGATAATCGCCCTTTATATGCA
NtPDS	ATGCTAACTAGCTGGAGACTCTTTCCTGCTTCGTTCACCAGGCCTAAGTACTGCCCAAGTGGATTTTATTGCAGGGCTGAGGATCAACTGAGTAGTATAAATGAACGTCAAAAGAAGAAGAAAGTGCTGATAGTAGGTTCAGGCTGGGCTGGCCTTGGAGCTGCTCACCATCTCTGCAAACAGGGCTTTGAGGTCGTTGTTCTTGAAGGTGGCTATGAATTTGGACCCAAAAATCAATCCCTAAGCCCTGACGATGTGGCTATTCGCGGTTTCTGGTATCCCTATCGAAATATATTTGAT

顺式元件 Cis-element	序列 Sequence （5′-3′）	数量 Number	功能 Function
CAAT-box	CAAAT	24	启动子和增强子区域的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions
GARE-motif	TCTGTTG	3	赤霉素响应元件 Gibberellin-responsive element
LTR	CCGAAA	2	参与低温响应的顺式作用元件 Cis-acting element involved in low-temperature responsiveness
TCT-motif	TCTTAC	2	光响应元件的组成部分 Components of a light responsive element
TATA-box	TACAAAA	47	转录起始点-30附近的核心启动子元件 Core promoter element around -30 of transcription start
TCA-element	CCATCTTTTT	1	参与水杨酸反应的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness
TGACG-motif	TGACG	2	参与茉莉酸甲酯（MeJA）响应的顺式调控元件 Cis-acting regulatory element involved in the MeJA-responsiveness
O2-site	GATGATGTGG	1	参与玉米醇溶蛋白代谢调控的顺式调控元件 Cis-acting regulatory element involved in zein metabolism regulation
MRE	AACCTAA	1	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
chs-CMA2a	TCACTTGA	1	光响应元件 Components of a light responsive element
ATCT-motif	AATCTAATCC	1	光响应保守DNA模块的组成部分 Components of a conserved DNA module involved in light responsiveness
CGTCA-motif	CGTCA	1	参与茉莉酸甲酯响应的顺式作用调控元件 Cis-acting regulatory element involved in the MeJA-responsiveness
GT1-motif	GGTTAA	1	光响应元件 Light responsive element