茶树CsTMFs的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1408

摘要/Abstract

摘要：

基于茶树全基因组数据，筛选鉴定茶树（Camellia sinensis（L.）O. Ktze.）TATA元件调控因子（TATA element modulatory factor，TMF）CsTMFs家族成员。克隆茶树叶片中CsTMFs编码区序列（coding sequence，CDS）全长。使用生物信息学方法对CsTMFs的保守结构域、基因结构、理化性质、蛋白质二级结构和系统发育关系进行分析。基于转录组数据进行组织特异性表达的分析，利用实时荧光定量聚合酶链式反应（real-time fluorescent quantitative PCR，qPCR）方法检测冷胁迫和干旱胁迫下CsTMFs在茶树叶片中的表达情况。结果表明，CsTMFs家族有2个CsTMF基因，序列CDS区长度分别为2 934和2 904 bp，均具有TMF-DNA-bd和TMF-TATA-bd完整保守结构域。CsTMF1和CsTMF2具有组织表达特异性，CsTMF1在花与茎中表达较高，CsTMF2在成熟叶与茎中表达较高。CsTMF1受冷胁迫诱导，CsTMF2受冷胁迫和干旱胁迫抑制。

关键词: 茶树, TMF, 基因克隆, 表达分析

Abstract:

Based on the whole genome data of tea plant（Camellia sinensis）, members of tea plant TATA element modulatory factor（CsTMF）gene family were screened and identified. The full-length coding sequence（CDS）of CsTMFs in the tea plant leaves was further cloned. The conserved domain, gene structure, physical and chemical properties, protein secondary structure and phylogenetic relationship of CsTMFs were analyzed by bioinformatics method. Tissue-specific expression was analyzed based on transcriptome data, and qPCR was used to detect the expressions of CsTMFs in the tea plant leaves under cold stress and drought stress. The result showed that there were two genes of CsTMF gene family in the tea plant. The lengths of CDS region in two CsTMF genes were 2 934 bp and 2 904 bp, respectively. There were complete conserved domains, TMF-DNA-bd and TMF-TATA-bd in both two CsTMF genes. CsTMF1 and CsTMF2 had tissue expression specificity. CsTMF1 was highly expressed in the flowers and stems, while CsTMF2 was highly expressed in the mature leaves and stems. The results of qPCR showed that CsTMF1 was induced by cold stress, while CsTMF2 was inhibited by cold stress and drought stress.

Key words: tea plant, TMF, gene cloning, expression analysis

孙明慧, 吴琼, 刘丹丹, 焦小雨, 王文杰. 茶树CsTMFs的克隆与表达分析[J]. 生物技术通报, 2023, 39(7): 151-159.

SUN Ming-hui, WU Qiong, LIU Dan-dan, JIAO Xiao-yu, WANG Wen-jie. Cloning and Expression Analysis of CsTMFs Gene in Tea Plant[J]. Biotechnology Bulletin, 2023, 39(7): 151-159.

图/表 13

表1 PCR 引物信息

Table 1 Primer sequences for PCR

用途Application	引物名称Primer name	引物序列Primer sequence（5'-3'）
实时荧光定量PCR qPCR	CsTMF1-qF	TCCATCCGTGACTCTCTTGCTGAG
	CsTMF1-qR	GCCTCCGCCTTAGTGCCTCTA
	CsTMF2-qF	CGACTGGCATCTCTGGAATCC
	CsTMF2-qR	GCCTCCGTCTTAATGCTTCTAACT
	Actin-qF	GCCATCTTTGATTGGAATGG
	Actin-qR	GGTGCCACAACCTTGATCTT
基因克隆Gene cloning	CsTMF1-F	ATGGCCTGGTTTAGCGGGAAAGTCTCTTTGG
	CsTMF1-R	TCATGCAGAGCCCATAGAAGAACCCATTACC
	CsTMF2-F	ATGGCCTGGTTCGGTGGGAAAGTCT
	CsTMF2-R	CTAAGACGAACTTAGTACCTGGATC

图1 CsTMFs蛋白的结构域分析

Fig. 1 Domain analysis of CsTMFs proteins

图2 CsTMFs扩增电泳图 M：5 000 bp marker

Fig. 2 CsTMFs amplification electropherogram

图3 CsTMFs保守基序（A）及基因结构（B）

Fig. 3 Conserved motifs（A）and gene structures（B）of CsTMFs members

表2 CsTMFs蛋白序列中10个保守基序的详细信息

Table 2 Detailed information of 10 motifs in the CsTMFs proteins

Motif	宽度Width	最可能序列Most possible match	结构域Domain
1	80	TARLQPSTVPDQSPITRRKSGFENGNLSRKLSSASSLGSMEESFFLQASLDSSDNFSERRNPGEMAMSPYFMKSMPPSAF	*
2	78	TMLVQALEELRQTLSRKEQQAVFREDMLRRDIEDLQKRYQESERRCEELVTQVPESTRPLLRQIEAMQETTGRRAEAW	*
3	40	MAWFGGKVSLGNFPDLAGAVNKISESVKNIEKNFDSALGF	*
4	80	MKMMETALQGAARQAQAKADEIAKLMNENENQKAVIEDLRRKSNEAEIESLREEYHQKVAALERKVYALTKERDTLRREQ	*
5	80	IRDSLAEELVKMTAQCEKLRVEAAILPGIRAELEALRRRHSAALELMGERDEELEELRADIVDLKEMYREQVNLLVNKIQ	TMF-TATA-bd
6	79	GLWPSGTDRKTJFEPVIAFMGHKGGESTVESIEKPELLEPPSSVEEKZEGENDRSTDFATEQTRPAEEVNZESSHMLPD	*
7	79	QAGMEVELPGFGFPTSKEIESAEEHFTDDLPDGPPPDEAAEMVSEPVSHENDTFGREVELNZQASDYEPDIKEQRVSSG	TMF-DNA-bd
8	80	VMAEGEELSKKQAAQESQIRKLRAQIRELEEEKKGLTTKLQVEENKVESIKRDKAETEKLLQETIEKHQAEJAVQKEFYT	*
9	80	QTLSRLNVLEAQISCLRAEQTQLSRSLEKERQRAAENRQEYLAAKEEADTHEGRVSQLEEEIRELRRKQKZELQEALMHR	*
10	66	TSALGPFENSELVEAKPGTNEVDQVEVGAPVPPESHNVINLHEISDEQKAZEEEIVENLSPVQAED	*

图4 CsTMFs蛋白二级结构预测 α-螺旋（蓝色）、延伸链（红色）、β-转角（绿色）和随机线圈（紫色）

Fig. 4 Secondary structure prediction of CsTMFs protein α-helix（blue）, extended strand（red）, β-turn（green）and random coil（purple）

图5 CsTMFs家族染色体定位

Fig. 5 Chromosome location of CsTMFs family

图6 STRING软件以水稻蛋白质数据库为参考预测CsTMFs蛋白互作网络模型

Fig. 6 Interaction network of CsTMFs based on Oryza sativa protein database by STRING software

图7 茶树CsTMFs系统进化树分析

Fig. 7 Phylogenetic tree analysis of CsTMFs from tea plant and other plant species

表3 不同物种TMF登录号

Table 3 Accession numbers of different species of TMF

物种名Species name	登录号Accession No.
水稻Oryza sativa Japonica Group	BAG90311.1
茭白Zizania palustris	KAG8086397
小麦Triticum aestivum	XP 044346450.1
大麦Hordeum vulgare subsp. Vulgar	BAJ99499
玉米Zea mays	XP 008656031.1

图8 茶树与其他植物TMF的多重序列比对紫色框内为TMF-DNA-bd保守结构域，黑色框内为TMF-TATA-bd保守结构域

Fig. 8 Multiple sequence alignment of tea plant TMF with other plants The conserved domain of TMF-DNA-bd is in purple box, and the conserved domain of TMF-TATA-bd is in black box

图9 CsTMF1与CsTMF2在茶树不同组织中的表达情况热图体现的log5（FPKM+1）值

Fig. 9 Expressions of CsTMF1 and CsTMF2 in different tissues of tea plant The log5（FPKM+1）value reflects in the heatmap

图10 CsTMFs在低温和干旱胁迫下的表达模式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. 10 Expression pattern of CsTMFs gene under drought stress and low temperature Different lowercase letters in figure indicate significant differences at different time of the same treatment（P<0.05）

参考文献 20

[1]	徐燕, 朱创, 邰玲玲, 等. 红茶化学成分及生理活性的研究进展[J]. 安徽农业大学学报, 2020, 47(5): 687-696.
	Xu Y, Zhu C, Tai LL, et al. Research progress on chemical constituents and physiological activities of black tea[J]. J Anhui Agric Univ, 2020, 47(5): 687-696.
[2]	陈思文, 康芮, 郭志远, 等. 茶树CsCML16基因的克隆及其低温胁迫下的表达分析[J]. 茶叶科学, 2021, 41(3): 315-326.
	Chen SW, Kang R, Guo ZY, et al. Cloning and expression analysis of CsCML16 in tea plants(Camellia sinensis)under low temperature stress[J]. J Tea Sci, 2021, 41(3): 315-326.
[3]	刘声传, 陈亮. 茶树耐旱机理及抗旱节水研究进展[J]. 茶叶科学, 2014, 34(2): 111-121.
	Liu SC, Chen L. Research advances on the drought-resistance mechanism and strategy of tea plant[J]. J Tea Sci, 2014, 34(2): 111-121.
[4]	Zhou CZ, Zhu C, Fu HF, et al. Genome-wide investigation of superoxide dismutase(SOD)gene family and their regulatory miRNAs reveal the involvement in abiotic stress and hormone response in tea plant(Camellia sinensis)[J]. PLoS One, 2019, 14(10): e0223609.
[5]	Garcia JA, Ou SH, Wu F, et al. Cloning and chromosomal mapping of a human immunodeficiency virus 1 TATA element modulatory factor[J]. Proc Natl Acad Sci USA, 1992, 89(20): 9372-9376. pmid: 1409643
[6]	Elkis Y, Bel S, Lerer-Goldstein T, et al. Testosterone deficiency accompanied by testicular and epididymal abnormalities in TMF^-/- mice[J]. Mol Cell Endocrinol, 2013, 365(1): 52-63. doi: 10.1016/j.mce.2012.09.003 pmid: 23000399
[7]	Fridmann-Sirkis Y, Siniossoglou S, Pelham HRB. TMF is a golgin that binds Rab6 and influences Golgi morphology[J]. BMC Cell Biol, 2004, 5: 18. pmid: 15128430
[8]	Abrham G, Volpe M, Shpungin S, et al. TMF/ARA160 downregulates proangiogenic genes and attenuates the progression of PC3 xenografts[J]. Int J Cancer, 2009, 125(1): 43-53. doi: 10.1002/ijc.24277 pmid: 19330832
[9]	徐艳. 水稻OsSKIPa互作蛋白OsTMF和OsARID3在抗逆和发育中的功能研究[D]. 武汉: 华中农业大学, 2015.
	Xu Y. Study on the function of rice OsSKIPa interaction proteins OsTMF and OsARID3 in stress resistance and development[D]. Wuhan: Huazhong Agricultural University, 2015.
[10]	Xu Y, Hu D, Hou X, et al. OsTMF attenuates cold tolerance by affecting cell wall properties in rice[J]. New Phytol, 2020, 227(2): 498-512. doi: 10.1111/nph.16549 pmid: 32176820
[11]	Miura K, Furumoto T. Cold signaling and cold response in plants[J]. Int J Mol Sci, 2013, 14(3): 5312-5337. doi: 10.3390/ijms14035312 pmid: 23466881
[12]	Chinnusamy V, Zhu JH, Zhu JK. Cold stress regulation of gene expression in plants[J]. Trends Plant Sci, 2007, 12(10): 444-451. doi: 10.1016/j.tplants.2007.07.002 pmid: 17855156
[13]	Latijnhouwers M, Gillespie T, Boevink P, et al. Localization and domain characterization of Arabidopsis golgin candidates[J]. J Exp Bot, 2007, 58(15/16): 4373-4386. doi: 10.1093/jxb/erm304 URL
[14]	Li YY, Wang XW, Ban QY, et al. Comparative transcriptomic analysis reveals gene expression associated with cold adaptation in the tea plant Camellia sinensis[J]. BMC Genomics, 2019, 20(1): 624. doi: 10.1186/s12864-019-5988-3
[15]	唐磊, 肖罗丹, 黄伊凡, 等. 茶树CsMGTs基因的克隆及其镁转运功能分析[J]. 茶叶科学, 2021, 41(6): 761-776.
	Tang L, Xiao LD, Huang YF, et al. Cloning of CsMGTs gene from tea plant and analysis of its magnesium transport function[J]. J Tea Sci, 2021, 41(6): 761-776.
[16]	Wu Q, Tong W, Zhao HJ, et al. Comparative transcriptomic analysis unveils the deep phylogeny and secondary metabolite evolution of 116 Camellia plants[J]. Plant J, 2022, 111(2): 406-421. doi: 10.1111/tpj.v111.2 URL
[17]	Li P, Guo WZ. Genome-wide characterization of the Rab gene family in Gossypium by comparative analysis[J]. Bot Stud, 2017, 58(1): 26. doi: 10.1186/s40529-017-0181-y URL
[18]	裴惠娟, 张满效, 安黎哲. 非生物胁迫下植物细胞壁组分变化[J]. 生态学杂志, 2011, 30(6): 1279-1286.
	Pei HJ, Zhang MX, An LZ. Changes of plant cell wall components under abiotic stresses: a review[J]. Chin J Ecol, 2011, 30(6): 1279-1286.
[19]	陈媛媛. 利用CRISPR/Cas9技术初步解析水稻纤维素合酶(OsCESA4和OsCESA7)P-CR区的功能[D]. 武汉: 华中农业大学, 2018.
	Chen YY.The function of P-Cr region of rice cellulose synthase(OsCESA4 and OSCESA7)was preliminarily analyzed by CRISPR/Cas 9 technology.[D]. Wuhan: Huazhong Agricultural University, 2018.
[20]	Liu HH, Ma Y, Chen N, et al. Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice[J]. Plant Cell Environ, 2014, 37(5): 1144-1158.. doi: 10.1111/pce.2014.37.issue-5 URL