Zhou Pei-Na, Zhu Zai-Biao, Xiong Lei, Zhang Ying, Chen Peng, Tong Huang-Jin, Fei Cheng-Hao
Nanjing Research Institute for Comprehensive Utilization of Wild Plants,AU China Federation of Supply and Marketing Cooperatives Nanjing 210042, China.
Institute of Chinese Medicinal Materials, Nanjing Agricultural University Nanjing 210095, China.
Zhongguo Zhong Yao Za Zhi. 2025 May;50(10):2658-2673. doi: 10.19540/j.cnki.cjcmm.20250217.103.
Based on whole-genome identification of the TPS gene family in Perilla frutescens and screening, cloning, bioinformatics, and expression analysis of the synthetic enzyme for the insect-resistant component germacrene D, this study lays the foundation for understanding the biological function of the TPS gene family and the insect resistance mechanism in P. frutescens. This study used bioinformatics tools to identify the TPS gene family of P. frutescens based on its whole genome and predicted the physicochemical properties, systematic classification, and promoter cis-elements of the proteins. The relative content of germacrene D was detected in both normal and insect-infested leaves of P. frutescens, and the germacrene D synthase was screened and isolated. Gene cloning, bioinformatics analysis, and expression profiling were then performed. The results showed that a total of 99 TPS genes were identified in the genome, which were classified into the TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g subfamilies. Conserved motif analysis showed that the TPS in P. frutescens has conserved structural characteristics within the same subfamily. Promoter cis-element analysis predicted the presence of light-responsive elements, multiple hormone-responsive elements, and stress-responsive elements in the TPS family of P. frutescens. Transcriptome data revealed that most of the TPS genes in P. frutescens were highly expressed in the leaves. GC-MS analysis showed that the relative content of germacrene D significantly increased in insect-damaged leaves, suggesting that it may act as an insect-resistant component. The germacrene D synthase gene was screened through homologous protein binding gene expression and was found to belong to the TPS-a subfamily, encoding a 64.89 kDa protein. This protein was hydrophilic, lacked a transmembrane structure and signal peptide, and was predominantly expressed in leaves, with significantly higher expression in insect-damaged leaves compared to normal leaves. In vitro expression results showed that germacrene D synthase tended to form inclusion bodies. Molecular docking showed that farnesyl pyrophosphate(FPP) fell into the active pocket of the protein and interacted strongly with six active sites. This study provides a foundation for further research on the biological functions of the TPS gene family in P. frutescens and the molecular mechanisms underlying its insect resistance.
基于紫苏全基因组中TPS基因家族的鉴定以及抗虫成分吉马烯D合成酶的筛选、克隆、生物信息学分析和表达分析，本研究为了解TPS基因家族的生物学功能以及紫苏的抗虫机制奠定了基础。本研究利用生物信息学工具基于紫苏全基因组鉴定其TPS基因家族，并预测了蛋白质的理化性质、系统分类和启动子顺式元件。检测了紫苏正常叶片和虫害叶片中吉马烯D的相对含量，并筛选分离出吉马烯D合酶。随后进行了基因克隆、生物信息学分析和表达谱分析。结果表明，在基因组中共鉴定出99个TPS基因，它们被分为TPS-a、TPS-b、TPS-c、TPS-e/f和TPS-g亚家族。保守基序分析表明，紫苏中的TPS在同一亚家族内具有保守的结构特征。启动子顺式元件分析预测紫苏TPS家族中存在光响应元件、多种激素响应元件和胁迫响应元件。转录组数据显示，紫苏中的大多数TPS基因在叶片中高表达。气相色谱-质谱分析表明，虫害叶片中吉马烯D的相对含量显著增加，表明其可能作为一种抗虫成分。通过同源蛋白结合基因表达筛选出吉马烯D合酶基因，发现其属于TPS-a亚家族，编码一个64.89 kDa的蛋白质。该蛋白具有亲水性，缺乏跨膜结构和信号肽，主要在叶片中表达，与正常叶片相比，在虫害叶片中的表达显著更高。体外表达结果表明，吉马烯D合酶倾向于形成包涵体。分子对接表明，法呢基焦磷酸（FPP）落入该蛋白的活性口袋，并与六个活性位点强烈相互作用。本研究为进一步研究紫苏TPS基因家族的生物学功能及其抗虫分子机制提供了基础。
