茶树乙烯受体<i>ETR</i>基因家族的鉴定与表达分析

晏美红; 郑玉成; 侯炳豪; 陈雪津; 陈晓君; 叶乃兴

doi:10.19303/j.issn.1008-0384.2021.10.007

茶树乙烯受体ETR基因家族的鉴定与表达分析

doi: 10.19303/j.issn.1008-0384.2021.10.007

福建农林大学园艺学院/茶学福建省高校重点实验室，福建　福州　350002

基金项目: 中国乌龙茶产业协同创新中心专项（闽教科〔2015〕75号）；安溪茶叶重大科技创新专项（AX2021001）；福建农林大学大学生创新创业训练计划项目（20190389154）；福建张天福茶叶发展基金会科技创新基金项目（FJZTF01）

详细信息

作者简介:
晏美红（1998−），女，研究方向：茶树栽培育种与生物技术（E-mail：15960010793@163.com）

通讯作者:
叶乃兴（1963−），男，教授，研究方向：茶树栽培育种与品质化学（E-mail：ynxtea@126.com）

中图分类号: S 571.1
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- 被引次数: 0
出版历程
- 收稿日期: 2021-05-26
- 修回日期: 2021-08-30
- 网络出版日期: 2021-10-23
- 刊出日期: 2021-10-28

Identification and Expression Analysis of ETR Gene Family in Camellia sinensis

College of Horticulture, Fujian Agriculture and Forestry University/Key Laboratory of Tea Science at University in Fujian, Fuzhou, Fujian　350002, China

摘要

摘要: 目的鉴定并分析CsETR基因，预测其潜在分子功能，以期从分子水平了解乙烯受体在茶树生长过程中应对胁迫的作用机制。方法采用生物信息学分析方法鉴定ETR基因家族成员并预测其潜在分子功能，采用实时荧光定量分析法分析其在不同胁迫处理下的表达水平。结果从茶树基因组中确定了6个茶树ETR基因家族成员，均包含N端跨膜区、GAF区和组氨酸（His）激酶结构域；系统发育树分析显示其为两组，与乙烯受体的两个亚家族分别对应；茶树ETR基因家族中每个成员含有1～12个外显子。ETR家族基因在茶树果实和茎中表达量较高，且该家族表现出明显的组织表达特异性。实时荧光定量分析结果显示：低温处理下，6个基因的表达水平上调，其中ERS1-1基因的表达水平上调最为明显；在植物生长调节剂ABA、JA和GA处理下大部分基因表达水平上调，其中ERS1-3基因上调最为明显，而ETR2-2基因在ABA处理下表达水平呈缓慢下调趋势。结论共鉴定出6个CsETR基因家族成员，并预测分析了其潜在的分子功能，该基因家族成员在果实和茎中表达量较高，且低温能诱导CsETR基因的表达，其中ERS1-3基因在ABA、MeJA和GA的处理下表达量显著上调。
- 茶树 /
- ETR基因 /
- 植物生长调节剂 /
- 非生物胁迫 /
- 表达分析
Abstract: Objective CsETRs were identified and analyzed to predict the potential molecular functions involving the mechanism of ethylene receptor in response to stress in tea plants. Method Bioinformatics was used to identify members in the ETR family and predict their potential molecular functions. Real-time fluorescence quantitative analysis was employed for the expressions of the genes under stresses. Result Six ETRs were identified from the genome of tea plants. They all consisted of N-terminal transmembrane region, GAF region, and histidine (His) kinase domain. Phylogenetic tree analysis divided them into two groups. Each member of the ETR family contained 1-12 exons. Significantly differentiated in tissues, the expressions of ETRs were high in the fruits and stems. The fluorescence quantitative expressions of the 6 genes, especially ERS1-1, were upregulated to varying degrees when exposed to low temperature. Under the stress of plant growth regulators ABA, JA, or GA, most of the genes were upregulated, especially ERS1-3, but ETR2-2 downregulated slowly when treated by ABA. Conclusion On tea plants, 6 CsETRs in the family were identified with their potential molecular functions predicted and analyzed in this study. CsETRs were highly expressed in the fruits and stems that could be induced by low-temperature stress. Whereas ABA, MeJA, or GA could significantly upregulate the expression of ERS1-3.
- Camellia sinensis /
- ETR gene /
- plant growth regulators /
- abiotic stress /
- expression analysis

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图 1 茶树乙烯受体结构域

Figure 1. Ethylene receptor domain of tea plants

下载: 全尺寸图片幻灯片

图 2 茶树与其他植物蛋白的ETR基因系统进化树

注：Cs.茶树；At.拟南芥；Le.番茄。

Figure 2. Phylogenetic tree of ETRs in tea and other plant proteins

Note: Cs: Camellia sinensis; At: Arabidopsis thaliana; Le: Lycopersicon esculentum.

下载: 全尺寸图片幻灯片

图 3 CsETR基因家族结构

注：a.CsETR家族系统进化树；b.CsETR基因结构。

Figure 3. Structure of CsETR family

Note: a: phylogenetic tree of CsETR family; b: structure of CsETR.

下载: 全尺寸图片幻灯片

图 4 CsETR基因的保守基序分析

注：a.CsETR家族系统进化树；b.CsETR家族保守基序分析；c.基序logo分析。

Figure 4. Conservative motif analysis on CsETRs

Note: a: phylogenetic tree of CsETR family; b: conserved motif distribution of CsETR family; c: motif logo analysis.

下载: 全尺寸图片幻灯片

图 5 CsETR基因启动子顺势元件预测

Figure 5. Predicted cis-elements in promoter of CsETRs

下载: 全尺寸图片幻灯片

图 6 CsETR基因家族的组织表达谱

注：蓝色代表低表达，橙色代表高表达。

Figure 6. Expression patterns of CsETR family

Note: blue represents low expression, and red high expression.

下载: 全尺寸图片幻灯片

图 7 CsETR基因在低温、GA、MeJA、ABA处理下的表达谱

注：对同一个基因相同处理下不同时间点的表达量进行差异性分析（ “*”表示P＜0.05）

Figure 7. Expression profile of CsETRs under low-temperature, GA, MeJA, or ABA treatment

Note: Expressions of same gene under identical treatment at different time ("*" indicates P＜ 0.05).

下载: 全尺寸图片幻灯片

表 1 茶树CsETR家族成员引物序列

Table 1. Primer sequence of CsETRs in tea plants

基因名称 Gene name	上游引物 Forward primers （5′→3′）	下游引物 Reverse primer （5′→3′）
ERS1-1	AATGGCCTGCTGATGAGCTT	CACAGAAGATCCAGCAGGCA
ERS1-2	GAGGATGGCAGCCTTCAACT	AGTACATCCCTTGCCAAGCC
ERS1-3	GGCACCAGATTTACCTGCCT	GCTGAGCTTTCTGCAACACC
EIN4	AATTGTGCCGTTTGGATGCC	GAGTTTCTTGCCTGGCTTGC
ETR2-1	GCAAATCGCTTGATCGGCAT	CCATCATTGCGCTCTGCTTC
ETR2-2	ATAAAGGAAGCCGCTTGCCT	TCCCGGTTCAGAGATGCCTA
CsGAPDH	TTGGCATCGTTGAGGGTCT	CAGTGGGAACAGGAAAGC

下载: 导出CSV

表 2 茶树CsETR基因家族的序列特征

Table 2. Characteristics of CsETR sequences in tea plants

基因 Gene name	基因组编号 Genome ID	编码序列长度 CDS/bp	开放阅读框长度 ORF/aa	分子量 MW/kD	等电点 pI	平均亲水性 Grand average of hydropathicity	亚细胞定位 Subcellular localization
CsERS1-1	TEA017521	1845	614	68.63	6.09	0.162	质膜 Plasma membrane
CsERS1-2	TEA032252	1923	640	71.71	7.02	0.144	质膜 Plasma membrane
CsERS1-3	TEA012203	1932	643	71.89	6.67	0.250	质膜 Plasma membrane
CsEIN4	TEA025082	2292	763	85.56	7.27	0.014	质膜 Plasma membrane
CsETR2-1	TEA002824	2292	763	84.73	6.32	0.077	质膜 Plasma membrane
CsETR2-2	TEA020178	2292	763	85.56	7.57	0.054	质膜 Plasma membrane

下载: 导出CSV

参考文献(34)

[1]	HAZRA A, DASGUPTA N, SENGUPTA C, et al. Next generation crop improvement program: Progress and prospect in tea (Camellia sinensis (L.) O. Kuntze) [J]. Annals of Agrarian Science, 2018, 16(2): 128−135.
[2]	HUA J, MEYEROWITZ E M. Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana [J]. Cell, 1998, 94(2): 261−271. doi: 10.1016/S0092-8674(00)81425-7
[3]	WEN X, ZHANG C L, JI Y S, et al. Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN₂ carboxyl Terminus [J]. Cell Research, 2012, 22(11): 1613−1616. doi: 10.1038/cr.2012.145
[4]	HUA J, SAKAI H, NOURIZADEH S, et al. EIN₄ and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis [J]. The Plant Cell, 1998, 10(8): 1321−1332. doi: 10.1105/tpc.10.8.1321
[5]	冯斗, 张春发, 张颖. 香蕉乙烯受体基因cDNA的克隆及其表达分析 [J]. 热带作物学报, 2004, 25(1):6−10. doi: 10.3969/j.issn.1000-2561.2004.01.002 FENG D, ZHANG C F, ZHANG Y. Ublished by Evert (2001). Slot Northern hybridization with the labeled probe of PGCHI indicated [J]. Chinese Journal of Tropical Crops, 2004, 25(1): 6−10.(in Chinese) doi: 10.3969/j.issn.1000-2561.2004.01.002
[6]	李丽, 孙健, 易萍, 等. 芒果乙烯受体ETR1和ERS1基因的克隆及序列分析 [J]. 南方农业学报, 2018, 49(6):1053−1060. doi: 10.3969/j.issn.2095-1191.2018.06.02 LI L, SUN J, YI P, et al. Cloning and sequence analysis of ethylene receptor ETR1 and ERS1 genes from Mango [J]. Journal of Southern Agriculture, 2018, 49(6): 1053−1060.(in Chinese) doi: 10.3969/j.issn.2095-1191.2018.06.02
[7]	陈志晟, 颜彦, 赵思涵, 等. 木薯ETR1基因克隆及表达分析 [J]. 南方农业学报, 2020, 51(1):19−26. doi: 10.3969/j.issn.2095-1191.2020.01.003 CHEN Z S, YAN Y, ZHAO S H, et al. Cloning and expression analysis of MeETR1 gene in cassava [J]. Journal of Southern Agriculture, 2020, 51(1): 19−26.(in Chinese) doi: 10.3969/j.issn.2095-1191.2020.01.003
[8]	王睿, 吴佳文, 毛仁俊, 陈国梁, 白朕卿. 丹参乙烯受体SmETR1基因的克隆及序列分析[J/OL]. 基因组学与应用生物学: 1−9[2021-05-06]. http://kns.cnki.net/kcms/detail/45.1369.Q.20210317.1532.002.html. WANG R, WU J W, ZHAO S H, et al. Cloning and sequence analysis of ethylene receptor SmETR1 gene from Salvia miltiorrhiza[J/OL]. Genomics and Applied Biology, 1-9[2021-05-06]. http://kns.cnki.net/kcms/detail/45.1369.Q.20210317.1532.002.html.
[9]	XIA E H, ZHANG H B, SHENG J, et al. The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis [J]. Molecular Plant, 2017, 10(6): 866−877. doi: 10.1016/j.molp.2017.04.002
[10]	WEI C L, YANG H, WANG S B, et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality [J]. PNAS, 2018, 115(18): E4151−E4158. doi: 10.1073/pnas.1719622115
[11]	WANG X C, FENG H, CHANG Y X, et al. Population sequencing enhances understanding of tea plant evolution [J]. Nature Communications, 2020, 11: 4447. doi: 10.1038/s41467-020-18228-8
[12]	CHEN J D, ZHENG C, MA J Q, et al. The chromosome-scale genome reveals the evolution and diversification after the recent tetraploidization event in tea plant [J]. Horticulture Research, 2020, 7: 63. doi: 10.1038/s41438-020-0288-2
[13]	ZHANG Q J, LI W, LI K, et al. The chromosome-level reference genome of tea tree unveils recent bursts of non-autonomous LTR retrotransposons in driving genome size evolution [J]. Molecular Plant, 2020, 13(7): 935−938. doi: 10.1016/j.molp.2020.04.009
[14]	陈丹, 俞滢, 岳川, 等. 茶树△12-脂肪酸去饱和酶基因FAD2和FAD6的克隆与表达分析 [J]. 茶叶科学, 2017, 37(6):541−550. doi: 10.3969/j.issn.1000-369X.2017.06.001 CHEN D, YU Y, YUE C, et al. Cloning and expression analysis of △12-fatty acid desaturase in tea plants [J]. Journal of Tea Science, 2017, 37(6): 541−550.(in Chinese) doi: 10.3969/j.issn.1000-369X.2017.06.001
[15]	王鹏杰, 郑玉成, 林浥, 等. 茶树GRF基因家族的全基因组鉴定及表达分析 [J]. 西北植物学报, 2019, 39(3):413−421. WANG P J, ZHENG Y C, LIN Y, et al. Genome-wide identification and expression analysis of GRF gene family in Camellia sinensis [J]. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39(3): 413−421.(in Chinese)
[16]	XIA E H, LI F D, TONG W, et al. Tea Plant Information Archive: A comprehensive genomics and bioinformatics platform for tea plant [J]. Plant Biotechnology Journal, 2019, 17(10): 1938−1953. doi: 10.1111/pbi.13111
[17]	SCHULTZ J, MILPETZ F, BORK P, et al. SMART, a simple modular architecture research tool: Identification of signaling domains [J]. PNAS, 1998, 95(11): 5857−5864. doi: 10.1073/pnas.95.11.5857
[18]	MARCHLER-BAUER A, DERBYSHIRE M K, GONZALES N R, et al. CDD: NCBI's conserved domain database [J]. Nucleic Acids Research, 2015, 43(Database issue): D222−D226.
[19]	DUVAUD S, GABELLA C, LISACEK F, et al. Expasy, the Swiss bioinformatics resource portal, as designed by its users [J]. Nucleic Acids Research, 2021, 49(W1): W216−W227. doi: 10.1093/nar/gkab225
[20]	HORTON P, PARK K J, OBAYASHI T, et al. WoLF PSORT: Protein localization predictor [J]. Nucleic Acids Research, 2007, 35(Web Server issue): W585−W587.
[21]	NEWMAN L, DUFFUS A L J, LEE C. Using the free program MEGA to build phylogenetic trees from molecular data [J]. The American Biology Teacher, 2016, 78(7): 608−612. doi: 10.1525/abt.2016.78.7.608
[22]	HU B, JIN J P, GUO A Y, et al. GSDS 2.0: An upgraded gene feature visualization server [J]. Bioinformatics, 2015, 31(8): 1296−1297. doi: 10.1093/bioinformatics/btu817
[23]	BAILEY T L, BODEN M, BUSKE F A, et al. MEME Suite: Tools for motif discovery and searching [J]. Nucleic Acids Research, 2009, 37(Suppl_2): W202−W208.
[24]	陈雪津, 王鹏杰, 郑玉成, 等. 茶树BES1转录因子全基因组鉴定与分析 [J]. 西北植物学报, 2019, 39(5):876−885. CHEN X J, WANG P J, ZHENG Y C, et al. Genome-wide identification and analysis of BES1 transcription factor family in Camellia sinensis [J]. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39(5): 876−885.(in Chinese)
[25]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method [J]. Methods, 2001, 25(4): 402−408. doi: 10.1006/meth.2001.1262
[26]	TIEMAN D M, KLEE H J. Differential expression of two novel members of the tomato ethylene-receptor family [J]. Plant Physiology, 1999, 120(1): 165−172. doi: 10.1104/pp.120.1.165
[27]	郭呈宇, 柳俊, 李园磊, 等. 甜瓜果实成熟相关基因家族的全基因组鉴定及分析 [J]. 华北农学报, 2019, 34(2):35−43. doi: 10.7668/hbnxb.201750903 GUO C Y, LIU J, LI Y L, et al. Genome-wide identification and analysis of gene families related to fruit ripening of melon [J]. Acta Agriculturae Boreali-Sinica, 2019, 34(2): 35−43.(in Chinese) doi: 10.7668/hbnxb.201750903
[28]	WATANABE H, SAIGUSA M, SHU H S, et al. Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments [J]. Journal of Experimental Botany, 2004, 55(399): 1145−1148. doi: 10.1093/jxb/erh110
[29]	XIE C, ZHANG Z G, ZHANG J S, et al. Spatial expression and characterization of a putative ethylene receptor protein NTHK₁ in tobacco [J]. Plant and Cell Physiology, 2002, 43(7): 810−815. doi: 10.1093/pcp/pcf095
[30]	GALLIE D R. Ethylene receptors in plants-why so much complexity? [J]. F1000Prime Reports, 2015, 7: 39.
[31]	ZHOU D B, KALAITZIS P, MATTOO A K, et al. The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues [J]. Plant Molecular Biology, 1996, 30(6): 1331−1338. doi: 10.1007/BF00019564
[32]	WURIYANGHAN H, ZHANG B, CAO W H, et al. The ethylene receptor ETR2 delays floral transition and affects starch accumulation in rice [J]. The Plant Cell, 2009, 21(5): 1473−1494. doi: 10.1105/tpc.108.065391
[33]	苏丽艳. 番茄SlETR6基因的克隆及非生物胁迫下的表达分析 [J]. 华北农学报, 2019, 34(1):19−25. doi: 10.7668/hbnxb.201751117 SU L Y. Cloning and expression analysis of ethylene receptor gene SlETR6 in Solanum lycopersicum under abiotic stress [J]. Acta Agriculturae Boreali-Sinica, 2019, 34(1): 19−25.(in Chinese) doi: 10.7668/hbnxb.201751117
[34]	黄静丽. 甘蔗乙烯受体基因SoERS1表达、转化及启动子序列的研究[D]. 南宁: 广西大学, 2013. HUANG J L. Study on sugarcane ethylene receptor gene SoERS1Expression, genetic transformation and promoter sequences[D]. Nanning: Guangxi University, 2013. (in Chinese)