茶树CsTDIFL基因的克隆、表达特性分析及其对氨基酸合成的调控

doi:10.13560/j.cnki.biotech.bull.1985.2025-0918

摘要/Abstract

摘要：

目的探究茶树TDIF信号肽对茶树氮代谢及氨基酸积累的调控作用，为进一步研究TDIF多肽影响茶树生理品质和氨基酸代谢的分子机制奠定基础。方法以茶树品种‘保靖黄金茶2号’为材料，克隆CsTDIFL基因并进行生物信息学分析；利用实时荧光定量PCR（RT-qPCR）检测CsTDIFL及其受体基因CsTDR在不同组织中的表达模式及外源TDIF多肽处理下的表达动态。结合HPLC和酶活性测定分析TDIF对氨基酸含量及氮代谢酶活性的影响。通过反义寡核苷酸（asODN）抑制CsTDIFL表达，验证其功能。结果 CsTDIFL编码1个含典型CLE基序和信号肽的胞外蛋白，在嫩叶中高表达，其受体基因CsTDR在茎中富集。外源TDIF处理可在4 h内诱导CsTDIFL和CsTDR基因显著上调，随后二者相对表达量显著下调并逐渐趋于处理前水平。10 μmol/L TDIF处理显著抑制茶氨酸与谷氨酸积累，并降低GS/GOGAT活性。经asODN处理24 h后，CsTDIFL转录水平较对照组下调约78.24%，而茶氨酸、谷氨酸含量显著升高约23.19%和34.53%，GS/GOGAT活性也显著升高。结论外源TDIF处理显著抑制GS/GOGAT活性及茶氨酸与谷氨酸积累，而瞬时沉默CsTDIFL则显著提升上述氨基酸含量与氮代谢关键酶活性，推测茶树TDIF信号可能通过抑制GS和GOGAT活性，减少谷氨酸供应，从而抑制茶氨酸等氨基酸的积累。

关键词: 茶树, CsTDIFL, 基因克隆, 氨基酸合成, 氮代谢, 组织表达分析, 生物信息学分析

Abstract:

Objective This study is aimed to investigate the regulatory role of the TDIF signaling peptide in nitrogen metabolism and amino acid accumulation in tea plants, thereby laying a foundation for further research into the molecular mechanisms of TDIF peptide influencing tea physiological quality and amino acid metabolism. Method Using the tea cultivar ‘Baojing Huangjincha 2’ as plant material, the CsTDIFL gene was cloned and analyzed by bioinformatics. Tissue-specific expression profiles of CsTDIFL and its receptor gene CsTDR, along with their expression dynamics under exogenous TDIF peptide treatment, were examined using quantitative real-time polymerase chain reaction (RT-qPCR). High-performance liquid chromatography (HPLC) and enzyme activity assays were applied to evaluate the effects of TDIF on amino acid content and key nitrogen metabolism enzymes. Functional validation was validated through transient suppression of CsTDIFL expression with antisense oligonucleotides (asODNs). Result CsTDIFL encoded an extracellular protein containing a typical CLE motif and a signal peptide, with high expression in young leaves, whereas CsTDR expression was predominant in stems. Exogenous TDIF treatment induced significant up-regulation of both CsTDIFL and CsTDR within 4 hours, followed by a marked decline toward baseline levels. Treatment with 10 μmol/L TDIF notably suppressed theanine and glutamate accumulation and reduced glutamine synthetase (GS) and glutamate synthase (GOGAT) activities. After 24 h of asODN interference, CsTDIFL transcript levels decreased by approximately 78.24% compared with the control, while theanine and glutamate contents increased by about 23.19% and 34.53%, respectively, accompanied by a significant increase in GS/GOGAT activities. Conclusion Exogenous TDIF treatment significantly inhibites GS/GOGAT activity and the accumulation of theanine and glutamate, whereas transient silencing of CsTDIFL enhances amino acid levels and key nitrogen metabolism enzyme activities. This supports the conclusion that the TDIF signal negatively regulates nitrogen metabolism in tea plants, likely through suppression of GS and GOGAT activities, thereby limiting glutamate supply and theanine biosynthesis.

Key words: tea plant, CsTDIFL, gene cloning, amino acids synthesis, nitrogen metabolism, tissue expression analysis, bioinformatic analysis

金旋, 向芬, 戴翠婷, 杨辉, 李赛君, 刘红艳, 钱思维, 蔺万煌, 李维. 茶树CsTDIFL基因的克隆、表达特性分析及其对氨基酸合成的调控[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0918.

JIN Xuan, XIANG Fen, DAI Cui-ting, YANG Hui, LI Sai-jun, LIU Hong-yan, QIAN Si-wei, LIN Wan-huang, LI Wei. Cloning and Expression Analysis of CsTDIFL and Its Regulation on Amino Acid Synthesis in Tea Plants （Camellia sinensis）[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0918.

图/表 6

图1 茶树根、茎、叶

Fig. 1 Root, stem, and leaf of the tea plant (Camellia sinensis L.)

图2 CsTDIFL基因的克隆及同源性和系统进化分析A：CsTDIFL蛋白与拟南芥CLE家族氨基酸序列比对分析；B：CsTDIFL蛋白与拟南芥CLE家族蛋白系统发育树及Motif分析

Fig. 2 Cloning, sequence homology and phylogenetic analysis of the CsTDIFL geneA:Amino acid sequence alignment analysis of CsTDIFL protein with the Arabidopsis CLE family. B:Phylogenetic tree and motif analysis of CsTDIFL protein and the Arabidopsis CLE family proteins

图3 CsTDIFL和CsTDR在茶树不同组织间的表达水平**表示极显著差异（P<0.01）

Fig. 3 Expressions of CsTDIFL and CsTDR in different tissues of tea plant** indicates extremely significant difference (P<0.01)

图4 茶树CsTDIFL及CsTDR对外源TDIF多肽激素的响应A‒C分别表示茶树叶、茎、根样品在TDIF多肽激素0 μmol/L（MOCK）、1 μmol/L和10 μmol/L处理下CsTDIFL和CsTDR的相对表达量；横坐标1‒24 h表示取样时间，处理前记为0 h；图中数据（A‒C）表示3个生物重复的平均值±SD。*表示显著性差异（P<0.05），**表示极显著性差异（P<0.01）。下同

Fig. 4 Responses of CsTDIFL and CsTDR to exogenous TDIF peptide in tea plantA‒C refer to the relative expressions of CsTDIFL and CsTDR in tea tree leaf, stem, and root samples, respectively, under treatments of 0 (MOCK), 1 μmol/L, and 10 μmol/L TDIF polypeptide hormone. The horizontal axis (1‒24 h) indicates the sampling time, with the pre-treatment time denoted as 0. The data in the figure (Panel A‒C) are the mean±SD of three biological replicates. * indicates a significant difference (P<0.05), and ** indicates a highly significant difference (P<0.01). The same below

图5 TDIF处理期间氨基酸含量及氮代谢关键酶活的动态变化A：TDIF处理24 h主要游离氨基酸的积累情况；B：TDIF处理0、1、4、12和24 h氮代谢关键酶活的动态变化

Fig. 5 Dynamics of amino acid contents and key nitrogen metabolic enzyme activities under TDIF treatmentA: Accumulation of major free amino acids after 24 h of TDIF treatment. B: Dynamic changes in key nitrogen metabolism enzyme activities under TDIF treatment at 0, 1, 4, 12, and 24 h

图6 反义寡核苷酸抑制CsTDIFL对茶氨酸含量的影响A：asODN处理24 h CsTDIFL表达量；B：asODN处理24 h茶氨酸和谷氨酸含量；C：asODN处理24 h GOGAT/GS酶活性

Fig. 6 Effects of antisense oligonucleotide inhibiting CsTDIFL on theanine contentA: CsTDIFL expressions after 24 h asODN treatment. B: Theanine and glutamate contents after 24 h of asODN treatment. C: GOGAT/GS enzyme activity after 24 h of asODN treatment

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