茶树TRB基因家族鉴定及表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0048

摘要/Abstract

摘要：

目的端粒结合蛋白（telomere repeat binding, TRB）是一类端粒双链DNA结合蛋白，对植物生长发育起着重要作用。通过鉴定茶树［Camellia sinensis（L.）O. Kuntze］TRB基因家族，克隆关键基因CsTRB1并解析其分子特性，研究CsTRBs在非生物胁迫下的表达模式，为揭示茶树TRB的功能提供分子基础。方法对茶树CsTRB基因家族进行全基因组鉴定，通过生物信息学分析保守基序、基因结构、染色体定位、基因共线性以及顺式作用元件，预测蛋白质理化性质及结构。以‘碧香早’为材料，克隆CsTRB1基因CDS序列。结合转录组数据和RT-qPCR对CsTRB基因家族成员在不同组织部位、低温胁迫和激素处理下的表达模式进行分析。结果鉴定出7个CsTRBs基因，成功克隆获得CsTRB1基因，其编码区全长885 bp，编码295个氨基酸，包含典型的TRB结构域。家族成员分布在6条染色体上。根据系统发育分析，将CsTRBs分为2个亚家族。共线性分析表明，CsTRB基因在茶树基因组内发生了基因复制事件，茶树TRB家族与拟南芥（Arabidopsis thaliana）的TRB家族之间有7对直系同源基因。RT-qPCR结果表明，在低温胁迫下，CsTRB1、CsTRB2、CsTRB6、CsTRB7显著下降，CsTRB3、CsTRB4、CsTRB5显著上升。除CsTRB5外，茶树CsTRB家族成员皆在ABA处理下高度表达。CsTRB1、CsTRB4、CsTRB5的表达量受到IAA处理的诱导而显著上调。结论 CsTRBs基因的表达可能受光、低温、激素等顺式作用元件影响，在茶树的生长发育和非生物胁迫响应中发挥重要作用。CsTRB1的成功克隆及其分子特性解析，为后续蛋白互作和功能验证研究提供了关键材料。

关键词: 茶树, TRB家族, 生物信息学, 系统进化, 顺式作用元件, 表达分析, 非生物胁迫, 全基因组鉴定

Abstract:

Objective Telomere repeat-binding (TRB) proteins, a class of double-stranded telomeric DNA-binding proteins, play crucial roles in plant growth and development. This study is aimed to identify the TRB gene family in tea plant [Camellia sinensis (L.) O. Kuntze], clone a key gene CsTRB1 and characterize its molecular properties, and investigate the expression patterns of CsTRBs under abiotic stresses. These findings may establish a molecular foundation for elucidating the functional mechanisms of TRB genes in C. sinensis. Method Genome-wide identification of CsTRB gene family was performed, with bioinformatics analyses of conserved motifs, gene structures, chromosomal localization, gene collinearity, and cis-acting elements, as well as predicting. Proteins' physicochemical properties and structures were predicted. Using 'Bixiangzao' as experimental material, the CDS sequence of CsTRB1 was cloned. Transcriptomic data and RT-qPCR were combined to analyze expression patterns of CsTRB family members across tissues, under low-temperature stress, and following hormone treatments. Result Seven CsTRB genes were identified. CsTRB1 was successfully cloned, containing an 885 bp coding sequence encoding 295 amino acids with a typical TRB domain. Family members were distributed across six chromosomes. CsTRBs were classified into two subfamilies via phylogenetic analysis. Collinearity analysis revealed gene duplication event of CsTRB gene within the tea genome and identified seven orthologous gene pairs between tea TRB family and Arabidopsis thalianaTRB family. RT-qPCR demonstrated that under low-temperature stress, the expressions of CsTRB1, CsTRB2, CsTRB6, and CsTRB7 significantly decreased, while the expressions of CsTRB3, CsTRB4, and CsTRB5 markedly increased. All CsTRB members except CsTRB5 showed high expression under ABA treatment. The expressions of CsTRB1, CsTRB4, and CsTRB5 was significantly upregulated by IAA treatment. Conclusion The expression of CsTRB gene may be regulated by cis-acting elements responsding to light, low temperature, and hormones, suggesting their critical roles in tea plant development and abiotic stress responses. The successful cloning and molecular characterization of CsTRB1 provide essential materials for subsequent protein interaction studies and functional validation.

Key words: Camellia sinensis, TRB gene family, bioinformatics, phylogenetic evolution, cis-acting element, expression analysis, abiotic stress, genome-wide identification

龚钰涵, 陈兰, 尚方慧子, 郝灵颖, 刘硕谦. 茶树TRB基因家族鉴定及表达模式分析[J]. 生物技术通报, 2025, 41(7): 214-225.

GONG Yu-han, CHEN Lan, SHANGFANG Hui-zi, HAO Ling-ying, LIU Shuo-qian. Identification and Expression Profile Analysis of the TRB Gene Family in Tea Plant[J]. Biotechnology Bulletin, 2025, 41(7): 214-225.

图/表 12

表1 引物信息

Table 1 Primer information

基因 Gene	正向引物 Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）
CsTRB1	TTTGAAGGAGCCTCGTGGG	GTTGCTGCCAACAGCCTTTC
CsTRB2	TGTTGATCTCAAGGACAAATGGAG	ATCCTGTTTAGGGGCAGGCT
CsTRB3	GGCACCTGCCAACTTCAAAC	CCCTCTGCCTTCCCTCTAGT
CsTRB4	TGGTTGTGGCAAAAACACCG	TGCCTCCTTAACGGCTTCAG
CsTRB5	AAAAGCAGAAGTGGACGGCT	CCATGTTCCGCCATTTGTCC
CsTRB6	CTGAATTTGCTCTTTCTCTCACCC	ATTTTGACCATTGGTGAGTGGG
CsTRB7	ATCCGGAATTTAGTGGCGTCT	CTCCATTTGTCCTTGAGATCAACA
CsGAPDH	TTGGCATCGTTGAGGGTCT	CAGTGGGAACACGGAAAGC
PCR-CsTRB1	ATGGTGGTCGCCTCCGGA	CTAAATCCAAATGCTACTGAAGGCT

表2 茶树CsTRBs蛋白物理化学性质

Table 2 Physicochemical properties of CsTRBs proteins in Camellia sinensis

基因 ID Gene ID	基因名 Gene name	氨基酸数Number of amino acids	蛋白质分子量 Molecular weight （Da）	等电点Theoretical pI	不稳定系数 Instability index	脂肪指数Aliphatic index	亲水性 Grand of hydropathicity	亚细胞定位 Subcellular localization prediction
CSS0029061	CsTRB1	294	32 011.53	8.56	34.83	82.52	-0.497	细胞核
CSS0034494	CsTRB2	302	33 233.12	9.52	46.42	82.28	-0.530	细胞核
CSS0012155	CsTRB3	300	32 813.69	9.56	49.24	81.57	-0.470	细胞核
CSS0030254	CsTRB4	280	30 857.84	9.16	40.08	69.79	-0.688	细胞核
CSS0035646	CsTRB5	264	29 439.74	9.71	37.99	81.40	-0.616	细胞核
CSS0046266	CsTRB6	264	29 439.74	9.71	37.99	81.40	-0.616	细胞核
CSS0022401	CsTRB7	302	33 234.06	9.52	46.42	80.99	-0.557	细胞核

图1 茶树CsTRB与其他TRB家族蛋白氨基酸序列比对图中红线标记为MYB结构域，黄线标记为组蛋白H1/5结构域，蓝线标记为卷曲螺旋结构域；Cs：茶树；At：拟南芥；Os：水稻；Zm：玉米

Fig. 1 Amino acid sequence alignment of CsTRB from tea plant with other TRB family proteinsThe MYB domain is marked with a red line, the histone H1/5 domain is marked with a yellow line, and the coil domain is marked with a blue line. Cs: Camellia sinensis. At: Arabidopsis thaliana. Os: Oryza sativa. Zm: Zea mays

图2 茶树、拟南芥、玉米和水稻TRB基因家族成员进化树

Fig. 2 Evolutionary tree of TRB gene family members in C. sinensis, A. thaliana, Z. mays, and O. sativa

图3 CsTRB1基因的克隆M：Marker 2 000；1：CsTRB1基因CDS序列扩增产物

Fig. 3 Cloning of the CsTRB1 geneM: Marker 2 000. 1: Amplified product of CDS sequence of CsTRB1

图4 茶树TRB家族基因基序、CDD及结构分析A：基于系统发育关系和结构域鉴定，将CsTRBs划分为2个支系；B：CsTRB蛋白中的保守基序组成，不同颜色的盒子表示不同的结构域；C：CsTRBs基因结构，蓝色矩形表示CDS或外显子，灰色线表示内含子

Fig. 4 Motif, CDD and structural analysis of TRB family genes in C. sinensisA: Identification based on phylogenetic relationships and domain, CsTRBs were classified into two subgroups. B: Composition of conserved motifs within CsTRB proteins. Boxes of different colors indicate distinct domains. C: Gene structures of CsTRBs. Blue rectangles denote CDS or exons, while gray lines indicate introns

图5 CsTRB基因的染色体定位和染色体间关联背景中的灰线显示了茶树基因组中所有的synsynblock，红线显示了synsynbnlor基因对

Fig. 5 Chromosomal localization and inter-chromosomal associations of CsTRB genesIn the background, gray lines indicate all the synsynblocks in the tea plant genome, while red lines indicate the synsynbnlor gene pairs

图6 茶树和拟南芥中TRB基因家族的共线性分析背景中的灰线表示茶树和拟南芥的共线性片段，蓝线表示共线性的TRB基因对

Fig. 6 Synteny analysis of TRB gene family between C. sinensis and A. thalianThe gray lines in the background depict syntenic blocks between the tea plant and Arabidopsis, while blue lines indicate syntenic TRB gene pairs

图7 CsTRB蛋白三维结构的同源性建模

Fig. 7 Homology modelling of the 3D structures of CsTRB proteins

图8 CsTRBs启动子区域顺式调控元件预测

Fig. 8 Prediction of cis-acting elements in the promoter region of CsTRBs

图9 CsTRB基因转录组表达谱A、C、D、E：分别表示CsTRB基因在不同组织、200 mmol/L NaCl、25% PEG和100 mmol/L MeJA胁迫下的表达分析，数据使用log2（FPKM+1）转换进行转换，红色方块表示基因被上调，蓝色方块表示基因被下调；B：茶树CsTRB基因在低温驯化下的表达分析（CK：25 ℃；Cold1-6 h：10 ℃，6 h；Cold1-7 d：昼/夜10 ℃/4 ℃，持续7 d；Cold2-7 d：昼/夜4 ℃/0 ℃ 7 d；ColdDA-7 d：在25 ℃，恢复7 d）

Fig. 9 Transcriptome expression profile of CsTRB genesA, C, D, and E: Indicate the expression analysis of CsTRB gene in different tissues, 200 mmol/L NaCl, 25% PEG and 100 mmol/L MeJA stress, the data were converted by log2 (FPKM+1), the red square indicates that the gene was up-regulated, and the blue square indicates that the gene was down-regulated. B: Expression analysis of CsTRB gene in tea plant under low temperature acclimation (CK: Non-acclimated at 25 ℃; Cold1-6 h: fully acclimated at 10 ℃ for 6 h; Cold1-7 d: 10 ℃/4 ℃ at day/night for 7 d; Cold2-7 d: 4 ℃/0 ℃ at day/night for 7 d; ColdDA-7 d: recovering under 25 ℃ for 7 d)

图10 CsTRB基因在不同非生物胁迫下的表达谱A：CsTRBs基因在低温胁迫下的表达谱；B：CsTRBs基因在100 μmol/L ABA处理下的表达谱；C：CsTRBs基因在100 μmol/L IAA处理下的表达谱。误差线代表3次独立生物学重复标准偏差；不同小写字母表示同一种处理不同时间点差异显著（P<0.05）

Fig. 10 Expression profile of CsTRB gene under different abiotic stressesA: Expression profile of CsTRBs gene under low temperature stress. B: Expression profile of CsTRBs gene under 100 μmol/L ABA treatment. C: Expression profile of the CsTRBs gene under 100 μmol/L IAA treatment. The error bars indicate the standard deviation of 3 independent biological replicates. Different lowercase letters indicate significant differences at different time points of the same treatment (P<0.05)

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