中国农业科学 ›› 2025, Vol. 58 ›› Issue (12): 2427-2438.doi: 10.3864/j.issn.0578-1752.2025.12.012

• 园艺 • 上一篇    下一篇

茶树‘英红9号’乙胺合成关键酶基因CsAlaDC转录因子的筛选

阮桥君(), 毛苗苗, 张媛媛, 林晓蓉, 陈忠正()   

  1. 华南农业大学食品学院,广州 510642
  • 收稿日期:2025-02-19 接受日期:2025-04-14 出版日期:2025-06-19 发布日期:2025-06-19
  • 通信作者:
    陈忠正,E-mail:
  • 联系方式: 阮桥君,E-mail:qiaojunruan@163.com
  • 基金资助:
    国家农业农村部农业科技创新条件提升建设工程(2020-440100-012846)

Screening of Transcription Factors for Key Enzyme Gene CsAlaDC Involved in Ethylamine Synthesis in Yinghong 9 (Camellia sinensis)

RUAN QiaoJun(), MAO MiaoMiao, ZHANG YuanYuan, LIN XiaoRong, CHEN ZhongZheng()   

  1. College of Food Science, South China Agricultural University, Guangzhou 510642
  • Received:2025-02-19 Accepted:2025-04-14 Published:2025-06-19 Online:2025-06-19

摘要:

【目的】茶氨酸是由底物乙胺和谷氨酸经茶氨酸合成酶催化合成。乙胺是茶树茶氨酸生成的主要限制因子,其由丙氨酸脱羧酶(CsAlaDC)催化丙氨酸脱羧而来。通过酵母单杂交从茶叶文库中筛选获得调控CsAlaDC的转录因子,从转录因子角度探究茶树体内乙胺合成调控的分子机制,为茶氨酸合成和后续开展茶树遗传改良提供理论基础和技术参考。【方法】以‘英红9号’茶树为材料,通过PCR克隆CsAlaDC启动子,以其为诱饵,利用酵母单杂交从文库中筛选获得候选转录因子,克隆候选转录因子的CDS,并回转验证。利用生物信息学方法对候选转录因子进行蛋白结构域、蛋白大小及可溶性分析,借助双荧光素酶试验探究候选转录因子对CsAlaDC的表达调控作用,并用亚细胞定位技术测定候选转录因子在细胞内的分布情况。【结果】克隆获得‘英红9号’茶树CsAlaDC启动子,包含真核生物基本的TATA-box和CAAT-box等启动子核心元件,I-box、G-Box、TCT-motif、TCCC-motif、Box4等光响应相关元件,CAT-box分生组织表达相关顺式作用元件,以及ABRE脱落酸响应元件、TC-rich repeats防御和压力反应顺式作用元件,还有MYB、MYC和WRKY等转录因子家族识别结合位点。以CsAlaDC启动子为诱饵进行酵母单杂交,从‘英红9号’cDNA文库中筛选获得17个结合蛋白,经初筛,获得5个候选转录因子,克隆CDS,并回转验证,获得3个转录因子:CsFBX、CsBBX和CsASR。双荧光素酶试验表明,CsBBX和CsASR对CsAlaDC具有转录激活作用,CsFBX无显著调控活性。亚细胞定位表明,CsFBX和CsBBX定位于细胞核,CsASR定位于细胞核和细胞质。【结论】从‘英红9号’cDNA文库中筛选获得3个调控乙胺合成关键酶基因CsAlaDC的转录因子:CsFBX、CsBBX和CsASR,均能结合CsAlaDC启动子,且CsBBX和CsASR对CsAlaDC具有转录激活作用。

关键词: 茶, 茶氨酸, 乙胺, 丙氨酸脱羧酶, 转录因子, 转录调控

Abstract:

【Objective】Theanine is synthesized from substrates ethylamine and glutamic acid catalyzed by theanine synthase. Ethylamine, the primary limiting factor in theanine biosynthesis in tea plants (Camellia sinensis), is generated via the decarboxylation of alanine catalyzed by alanine decarboxylase (CsAlaDC). This study aims to identify transcription factors regulating CsAlaDC through yeast one-hybrid screening of a tea plant cDNA library, thereby elucidating the molecular mechanisms underlying ethylamine biosynthesis regulation from a transcriptional perspective. The findings provide a theoretical foundation and technical references for advancing theanine biosynthesis and genetic improvement in tea plants.【Method】Using the Yinghong 9 tea cultivar, the promoter of CsAlaDC was cloned via PCR and employed as bait for yeast one-hybrid screening to identify candidate transcription factors from a cDNA library. Coding sequences (CDS) of candidate transcription factors were cloned and reversed to validate them. Bioinformatics analyses were conducted to characterize protein domains, molecular weights, and solubility of the candidates. Dual-luciferase assays were performed to assess transcriptional regulation of CsAlaDC by the candidate transcription factors, while subcellular localization experiments determined their cellular distribution.【Result】The CsAlaDC promoter cloned from the Yinghong 9 tea cultivar contained core eukaryotic promoter elements such as TATA-box and CAAT-box, along with light-responsive motifs (I-box, G-Box, TCT-motif, TCCC-motif, Box4), a meristem-specific CAT-box cis-element, stress-responsive elements (ABRE, TC-rich repeats), and binding sites for transcription factor families including MYB, MYC, and WRKY. Using the CsAlaDC promoter as bait, yeast one-hybrid screening of the Yinghong 9 cDNA library identified 17 potential binding proteins. After preliminary screening, five candidate transcription factors were selected for CDS cloning and reverse validation, ultimately confirming three transcription factors: CsFBX, CsBBX, and CsASR. Dual-luciferase assays demonstrated that CsBBX and CsASR significantly activated CsAlaDC expression, whereas CsFBX exhibited no regulatory effect. Subcellular localization revealed nuclear localization of CsFBX and CsBBX, while CsASR was localized in both the nucleus and cytoplasm.【Conclusion】Three transcription factors (CsFBX, CsBBX, and CsASR) regulating the ethylamine biosynthesis gene CsAlaDC were identified from the Yinghong 9 cDNA library. All three factors bound to the CsAlaDC promoter, with CsBBX and CsASR demonstrating transcriptional activation capabilities.

Key words: Camellia sinensis, theanine, ethylamine, alanine decarboxylase (CsAlaDC), transcription factors (TFs), transcriptional regulation