茶树海藻糖-6-磷酸合成酶（TPS）基因家族鉴定与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0252

摘要/Abstract

摘要：

【目的】海藻糖-6-磷酸合成酶（trehalose-6-phosphate synthase, TPS）是植物体内海藻糖生物合成通路中的关键酶。对茶树[Camellia sinensis（L.）O. Kuntze]TPS基因家族进行鉴定和分析，并研究其在逆境胁迫下的表达模式，为开展TPS基因功能验证提供基础。【方法】利用生物信息学方法鉴定茶树CsTPS基因家族成员，分析其编码的蛋白理化特性、保守基序与结构域、染色体定位、系统进化关系和启动子顺式作用元件等生物学信息，利用转录组数据结合RT-qPCR技术分析CsTPS家族成员在干旱、盐和低温胁迫下的表达模式。【结果】在茶树基因组中鉴定到16个TPS基因家族成员，系统发育分析将它们划分为Class I和Class II两个亚族。同一亚族成员具有相似的motif组成和基因结构。共线性与进化分析表明，CsTPS基因在茶树基因组内发生了基因复制事件，且它们在进化过程中经历了纯化选择作用。RT-qPCR结果显示，6个候选基因CsTPS3、CsTPS5、CsTPS6、CsTPS9、CsTPS11和CsTPS14均被PEG、NaCl和低温胁迫诱导，且表达模式与转录组结果一致。【结论】从茶树全基因组中系统鉴定出16个TPS家族成员。茶树CsTPS3、CsTPS5、CsTPS6、CsTPS9、CsTPS11和CsTPS14基因对干旱、盐和低温胁迫均有响应，但其敏感性水平各不相同。

关键词: 茶树, 海藻糖-6-磷酸合成酶基因, 系统进化, 表达模式, 非生物胁迫

Abstract:

【Objective】Trehalose-6-phosphate synthase（TPS）is a key enzyme in the trehalose biosynthetic pathway in plants. The TPS gene family of the tea plant[Camellia sinensis（L.）O. Kuntze]was identified, analyzed, and its expression pattern under adverse stress was studied. This study aims to provide a foundation for the functional validation of TPS genes.【Method】Utilizing biological information methods, we identified members of CsTPS gene family and analyzed the physical and chemical properties of their encoded protein, conserved motifs and domains, chromosomal location, phylogenetic relationships, promoter cis-acting elements, and other biological information. Subsequently, we employed transcriptome data in conjunction with RT-qPCR technology to investigate the expression patterns of CsTPS family members under drought, salt, and cold stress.【Result】The 16 TPS gene family members were identified in C. sinensis genome, which were classified into two subfamilies: Class I and Class II based on phylogenetic study. Members of the same subfamily presented similar motif composition and gene structure. The covariance and evolutionary studies revealed that CsTPS genes were duplicated within the tea plant genome and experienced purifying selection during the evolutionary process. RT-qPCR results revealed that the six candidate genes, CsTPS3, CsTPS5, CsTPS6, CsTPS9, CsTPS11, and CsTPS14, were all induced by PEG, NaCl, and low-temperature stress, and their expression patterns were similar with transcriptome information.【Conclusion】Sixteen TPS family members were identified systematically across the tea plant's genome. The CsTPS3, CsTPS5, CsTPS6, CsTPS9, CsTPS11, and CsTPS14 genes all are sensitive to drought, salt, and low-temperature stresses, though their sensitivity levels differed.

Key words: tea plant, trehalose-6-phosphate synthase gene, phylogeny, expression pattern, abiotic stress

刘丹丹, 王雷刚, 孙明慧, 焦小雨, 吴琼, 王文杰. 茶树海藻糖-6-磷酸合成酶（TPS）基因家族鉴定与表达分析[J]. 生物技术通报, 2024, 40(8): 152-163.

LIU Dan-dan, WANG Lei-gang, SUN Ming-hui, JIAO Xiao-yu, WU Qiong, WANG Wen-jie. Genome-wide Identification and Expression Pattern Profiling of the Trehalose-6-phosphate Synthase（TPS）Gene Family in Tea Plant（Camellia sinensis）[J]. Biotechnology Bulletin, 2024, 40(8): 152-163.

图/表 8

表1 Real-time PCR 引物信息

Table 1 Primer sequences for real-time PCR

基因名称Gene name	正向引物序列Forward primer sequence（5'-3'）	反向引物序列Reverse primer sequence（5'-3'）
β-actin	GCCATCTTTGATTGGAATGG	GGTGCCACAACCTTGATCTT
CsTPS3	TTCTCACAGAGAGTGATTGAGGTG	GGACTATGGAGAAAGAACCCCATT
CsTPS5	TTCTCACAGAGAGTGATTGAGGTG	GGACTATGGAGAAAGAACCCCATT
CsTPS6	TGTCTTTATTGTTAGTGGGAGGGG	CACAATTTCCTTCCACTCAAGGTC
CsTPS9	GCAAGATATGGAGAGAACTTGTGC	TGGGGCATTACAGTACCATCATAG
CsTPS11	GGTTACCTGTCTCTGCAGTTAGAA	CAAAGCTTTAGTAAGTGCCCTCTG
CsTPS14	GCCCTGATGATGATTTTGTATGGG	TTCAACTCTGACAGGTAGTGTACG

图1 拟南芥、舒茶早、云南野茶和浙江红山茶的TPS基因家族系统进化关系

Fig. 1 Phylogenetic relationship of TPS genes in A. thali-ana, C. sinensis ‘SCZ’, C. sinensis ‘DASZ’ and C. chekiangoleosa

图2 茶树中TPS基因的系统发育关系（A）、motif分析（B）与基因结构（C）

Fig. 2 Phylogenetic relationships（A）, motif compositions（B）and gene structure（C）of TPS genes in C. sinensis

图3 茶树TPS基因在染色体上的分布

Fig. 3 Distribution of TPS genes on the chromosome in C. sinensis

图4 茶树TPS基因家族启动子顺式作用元件预测

Fig. 4 Predicted cis-elements of promotors in TPS gene family of C. sinensis

图5 山茶属植物TPS基因共线性与进化选择压力分析 A：茶树TPS基因共线性分析，基因组内共线性用灰色线表示，CsTPS基因家族Class I与Class II亚族成员之间的共线性分别用橙色线和绿色线表示；B：舒茶早（SCZ）与浙江红山茶（CCH）之间TPS基因共线性分析；C：舒茶早与云南野茶（DASZ）之间TPS基因共线性分析；D：Ka/Ks分析；E：3种山茶属植物同源基因对Ks的密度分布

Fig. 5 Analysis of syntenic and Ka/Ks of TPS genes in Camellia plants A: Syntenic analysis of TPS genes in C. sinensis. The inner gray lines indicate all the collinearity relationships in the whole genome, and the inner orange and green lines indicate the collinearity relationships of Class I and Class II subfamilies, respectively. B: Syntenic analysis of TPS genes between C. sinensis ‘SCZ’ and C. chekiangoleosa. C: Syntenic analysis of TPS genes between C. sinensis ‘SCZ’ and C. sinensis ‘DASZ’. D: Analysis of Ka/Ks. E: Density distribution of Ks for paralogous gene pairs of the three species of Camellia plants

图6 茶树TPS基因转录组表达谱 A、C和D图分别表示CsTPS基因在不同组织、25% PEG胁迫和200 mmol/L NaCl胁迫下的表达模式；B：CsTPS基因在低温驯化下的表达模式（CK：白天25℃，晚上20℃，12 h光周期，70%湿度条件下生长1个月；CA1-6h：10℃处理6 h；CA1-7d：白天10℃，晚上4℃处理7 d；CA2-7d：白天4℃，晚上0℃处理7 d；DA-7d：白天25℃，晚上20℃处理7 d）；热图体现的log2（FPKM+1）值

Fig. 6 Transcriptome expression profiling of TPS genes in C. sinensis A, C and D graphs indicate the expression patterns of CsTPS genes in different tissues, 25% PEG stress and 200 mmol/L NaCl stress, respectively. B: Expression pattern of CsTPS gene under low temperature domestication（CK: 1 month of growth at 25℃ during the day, 20℃ at night, 12 h photoperiod and 70% humidity; CA1-6h: 6 h treatment at 10℃; CA1-7d: 10℃ during the day, 4℃ at night treatment for 7 d; CA2-7d: 4℃ during the day and 0℃ at night for 7 d; DA-7d: 25℃ during the day and 20℃ at night for 7 d）. The log2（FPKM+1）value reflects in the heatmap

图7 不同胁迫下CsTPSs基因的表达分析 A：20% PEG处理下CsTPSs基因的表达谱；B：200 mmol/L NaCl处理下CsTPSs基因的表达谱；C：4℃处理下CsTPSs基因的表达谱；D：24℃下CsTPSs基因生物钟表达谱；误差线代表3次独立生物学重复标准偏差，不同小写字母代表同一种处理不同时间点差异显著（P<0.05）

Fig. 7 Relative expressions of CsTPS genes under various stress A: Gene expression profiles of CsTPSs under 20% PEG. B: Gene expression profiles of CsTPSs under 200 mmol/L NaCl. C: Gene expression profiles of CsTPSs under 4℃ treatment. D: Circadian expression profiling of the CsTPS genes at 24℃. Error bars indicate the standard deviation of three independent biological replicates, different lowercase letters in indicate significant differences at different time of the same treatment（P<0.05）

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