Identification and Expression Profile Analysis of the TRB Gene Family in Tea Plant

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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

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)

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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