Cloning and Expression Analysis of E2 Ubiquitin-conjugating Enzyme Gene TkUBC2 in Taraxacum kok-saghyz Rodin

doi:10.7525/j.issn.1673-5102.2021.01.013

Abstract

Abstract:

In order to analyze the function of E2 ubiquitin-conjugating enzyme（UBC） in stress response and signal transduction， an UBC gene was cloned from Taraxacum kok-saghyz Rodin（TKS） line 1151 and named as TkUBC2. The ORF of this gene was 459 bp and encoded 152 aa. Sequence alignment analysis showed that UBC2 shared high level homology among different species， and TkUBC2 had more than 99% similarity with Lactuca sativa LsUBC2 and Helianthus annuus HaUBC2. qRT-PCR was used to analyze the expression pattern of TkUBC2in TKS. The results indicated that TkUBC2 was ubiquitously expressed in different tissues. Moreover， TkUBC2expression was down-regulated by treatments of PEG6000 simulated drought stress， mannitol mediated osmotic stress and plant hormone i.e. methyl jasmonate（MeJA）， abscisic acid（ABA） and ethylene（ET） respectively， but its expression was up-regulated by the treatments of NaCl salt stress and UV radiation respectively. The results of this study suggested that TkUBC2 was involved in response to various stress conditions， hormone signaling and DNA damage repair process， which lay a good foundation for further revealing the function of TkUBC2 in TKS.

Key words: Taraxacum kok-saghyz Rodin, TkUBC2, abiotic stress, expression analysis

CLC Number:

S576

Xiao-Xiao WANG, Bi QIN, Yu-Shuang YANG, Qiu-Hai NIE, Ji-Chuan ZHANG, Shi-Zhong LIU. Cloning and Expression Analysis of E2 Ubiquitin-conjugating Enzyme Gene TkUBC2 in Taraxacum kok-saghyz Rodin[J]. Bulletin of Botanical Research, 2021, 41(1): 98-106.

Figures/Tables 6

Table 1

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

References 37

1	Cui F，Liu L J，Zhao Q Z，et al.Arabidopsis ubiquitin conjugase UBC32 is an erad component that functions in brassinosteroid-mediated salt stress tolerance［J］.The Plant Cell，2012，24（1）：233-244.
2	Papaleo E，Casiraghi N，Arrigoni A，et al.Loop 7 of E2 enzymes：an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade［J］.PLoS One，2012，7（7）：e40786.
3	Schumacher F R，Wilson G，Day C L.The N-terminal extension of UBE2E ubiquitin-conjugating enzymes limits chain assembly［J］.Journal of Molecular Biology，2013，425（22）：4099-4111.
4	王金利，史胜青，贾利强，等.植物泛素结合酶E2功能研究进展［J］.生物技术通报，2010（4）：7-10.
	Wang J L，Shi S Q，Jia L Q，et al.Progress on functions of ubiquitin-conjugating enzyme（E2） in plants［J］.Biotechnology Bulletin，2010（4）：7-10.
5	Wang S，Li Q，Zhao L，et al.Arabidopsis UBC22，an E2 able to catalyze lysine-11 specific ubiquitin linkage formation，has multiple functions in plant growth and immunity［J］.Plant Science，2020，297：110520.
6	付学，唐勋，刘维刚，等.马铃薯StUBC12基因的生物信息学及表达特性分析［J］.农业生物技术学报，2020，28（5）：784-793.
	Fu X，Tang X，Liu W G，et al.Bioinformatics and expression characteristic analysis of StUBC12gene of potato（Solanum tuberosum）［J］.Journal of Agricultural Biotechnology，2020，28（5）：784-793.
7	徐东北，于月华，韩巧玲，等.大豆（Glycine max）GmDREB5互作蛋白GmUBC13的特性及功能［J］.中国农业科学，2014，47（18）：3534-3544.
	Xu D B，Yu Y H，Han Q L，et al.Characteristics and function of a GmDREB5-interacting protein GmUBC13 in soybean［J］.Scientia Agricultura Sinica，2014，47（18）：3534-3544.
8	Chung E，Cho C W，So H A，et al.Overexpression of VrUBC1，a mung bean E2 ubiquitin-conjugating enzyme，enhances osmotic stress tolerance in Arabidopsis［J］.PLoS One，2013，8（6）：e66056.
9	蔡佳文，金晓霞，于丽杰，等.龙葵E2泛素结合酶基因SorUBC克隆及表达特性分析［J］.东北农业大学学报，2016，47（11）：26-36.
	Cai J W，Jin X X，Yu L J，et al.Clone and expression pattern analysis of E2 ubiquitin-binding enzyme gene SorUBC in Solanum nigrum L.［J］.Journal of Northeast Agricultural University，2016，47（11）：26-36.
10	董轩名，蔡佳文，郑妍，等.蒜芥茄SsUBC基因的克隆及表达分析［J］.分子植物育种，2018，16（17）：5591-5600.
	Dong X M，Cai J W，Zheng Y，et al.Cloning and expression analysis of SsUBC gene in Solanum sisymbriifolium lam.［J］.Molecular Plant Breeding，2018，16（17）：5591-5600.
11	曾小玲，赵瑞丽，钟开勤，等.菜心BclUBE2基因的克隆与表达分析［J］.热带作物学报，2018，39（9）：1772-1777.
	Zeng X L，Zhao R L，Zhong K Q，et al.Cloning and expression analysis of ubiquitin-conjugating enzyme BclUBE2 gene in Brassica campestris L.ssp.chinensis var.utilis Tsen et lee［J］.Chinese Journal of Tropical Crops，2018，39（9）：1772-1777.
12	董晨，魏永赞，王弋，等.荔枝泛素结合酶基因（LcUBC12）的生物信息学及表达特性分析［J］.南方农业学报，2020，51（5）：1091-1097.
	Dong C，Wei Y Z，Wang Y，et al.Bioinformatics and expression characteristics analysis of Litchi chinesis Sonn ubiquitin-conjugating enzyme gene（LcUBC12）［J］.Journal of Southern Agriculture，2020，51（5）：1091-1097.
13	Xu Q Y，Yin S J，Ma Y，et al.Carbon export from leaves is controlled via ubiquitination and phosphorylation of sucrose transporter SUC2［J］.Proceedings of the National Academy of Sciences of the United States of America，2020，117（11）：6223-6230.
14	Zheng L，Chen Y J，Ding D，et al.Endoplasmic reticulum-localized UBC34 interaction with lignin repressors MYB221 and MYB156 regulates the transactivity of the transcription factors in Populus tomentosa［J］.BMC Plant Biology，2019，19（1）：97.
15	Watkins J F，Sung P，Prakash S，et al.The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation［J］.Genes & Development，1993，7（2）：250-261.
16	Koken M H M，Reynolds P，Jaspers-Dekker I，et al.Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6［J］.Proceedings of the National Academy of Sciences of the United States of America，1991，88（20）：8865-8869.
17	Cao Y，Dai Y，Cui S J，et al.Histone H₂B Monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis［J］.The Plant Cell，2008，20（10）：2586-2602.
18	Zwirn P，Stary S，Luschnig C，et al.Arabidopsis thaliana RAD6 homolog AtUBC2 complements UV sensitivity，but not N-end rule degradation deficiency，of Saccharomyces cerevisiae rad6 mutants［J］.Current Genetics，1997，32（5）：309-314.
19	Xu L，Ménard R，Berr A，et al.The E2 ubiquitin-conjugating enzymes，AtUBC1 and AtUBC2，play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana［J］.The Plant Journal，2009，57（2）：279-288.
20	Hoege C，Pfander B，Moldovan G L，et al.RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO［J］.Nature，2002，419（6903）：135-141.
21	Peng R H，Yao Q H，Xiong A S，et al.Ubiquitin-conjugating enzyme（E₂） confers rice UV protection through phenylalanine ammonia-lyase gene promoter unit［J］.Acta Botanica Sinica，2003，45（11）：1351-1358.
22	Song Y，Wang Y，Guo D D，et al.Selection of reference genes for quantitative real-time PCR normalization in the plant pathogen Puccinia helianthi Schw［J］.BMC Plant Biology，2019，19（1）：20.
23	Nong Q D，Yang Y C，Zhang M Y，et al.RNA-seq-based selection of reference genes for RT-qPCR analysis of pitaya［J］.FEBS Openbio，2019，9（8）：1403-1412.
24	Luo Z N，Iaffaldano B J，Zhuang X F，et al.Analysis of the first Taraxacum kok-saghyz transcriptome reveals potential rubber yield related SNPs［J］.Scientific Reports，2017，7（1）：9939.
25	刘明乾，吴绍华，田郎，等.橡胶草悬浮细胞遗传转化体系的建立［J］.热带生物学报，2018，9（2）：176-182.
	Liu M Q，Wu S H，Tian L，et al.Establishment of genetic transformation system for suspension cells of Taraxacum kok-saghyz Rodin［J］.Journal of Tropical Biology，2018，9（2）：176-182.
26	Benzle K，Cornish K.Improved axenic hydroponic whole plant propagation for rapid production of roots as transformation target tissue［J］.Plant Methods，2017，13：37.
27	尹丽娟，陈阳，刘沛，等.小麦泛素结合酶TaE2的表达分析及蛋白互作［J］.植物遗传资源学报，2014，15（1）：144-148，152.
	Yin L J，Chen Y，Liu P，et al.Expression pattern and protein interaction analysis of a wheat ubiquitin-conjugating enzyme TaE2［J］.Journal of Plant Genetic Resources，2014，15（1）：144-148，152.
28	安红强，范静，梁易，等.铁皮石斛泛素结合酶基因DoUBC24的克隆及表达分析［J］.生物技术通讯，2016，27（5）：643-648.
	An H Q，Fan J，Liang Y，et al.Cloning and expression analysis of ubiquitin-conjugating enzyme 24 in Dendrobium officinale Kimura et Migo［J］.Letters in Biotechnology，2016，27（5）：643-648.
29	Zhou G A，Chang R Z，Qiu L J.Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis［J］.Plant Molecular Biology，2010，72（4-5）：357-367.
30	Wan X R，Mo A Q，Liu S，et al.Constitutive expression of a peanut ubiquitin-conjugating enzyme gene in Arabidopsis confers improved water-stress tolerance through regulation of stress-responsive gene expression［J］.Journal of Bioscience and Bioengineering，2011，111（4）：478-484.
31	Baloglu M C，Patir M G.Molecular characterization，3D model analysis，and expression pattern of the CmUBC gene encoding the melon ubiquitin-conjugating enzyme under drought and salt stress conditions［J］.Biochemical Genetics，2014，52（1-2）：90-105.
32	李冬梅，李少旋，徐功勋，等.设施作物响应UV-B辐射的研究进展［J］.植物生理学报，2018，54（1）：36-44.
	Li D M，Li S X，Xu G X，et al.Research advances of plant response to UV-B radiation in greenhouse［J］.Plant Physiology Journal，2018，54（1）：36-44.
33	Graindorge S，Cognat V，Johann To Berens P，et al.Photodamage repair pathways contribute to the accurate maintenance of the DNA methylome landscape upon UV exposure［J］.PLoS Genetics，2019，15（11）：e1008476.
34	Mullenders L H F.Solar UV damage to cellular DNA：from mechanisms to biological effects［J］.Photochemical & Photobiological Sciences，2018，17（12）：1842-1852.
35	Schuch A P，Garcia C C M，Makita K，et al.DNA damage as a biological sensor for environmental sunlight［J］.Photochemical & Photobiological Sciences，2013，12（8）：1259-1272.
36	赖燕，徐波，林明，等.辣椒泛素交联酶cDNA的分离及其在UV-B作用下的表达分析［J］.福建农林大学学报：自然科学版，2008，37（2）：162-165.
	Lai Y，Xu B，Lin M，et al.Isolation of ubiquitin-conjugating enzyme cDNA from Capsicum annuum and analysis of its expression under UV-B stress［J］.Journal of Fujian Agriculture and Forestry University： Natural Science Edition，2008，37（2）：162-165.
37	Qin B.The function of Rad6 gene in Hevea brasiliensis extends beyond DNA repair［J］.Plant Physiology and Biochemistry，2013，66：134-140.

引物名称 Primer name	引物序列 Primer sequence(5′—3′)
TkUBC2-F	TCATTTTCCCGTTCACTTCC
TkUBC2-R	AAAAACTCATTTCACGCCATT
TkUBC2-QF	AGGTACGTTTAAGTTGACACTCCAG
TkUBC2-QR	AACATATGCTTCCATCTGCATAAAT
TkActin-F	GGAAGGATCTTTATGGGAAC
TkActin-R	CAGACACTATACTTCCTCTCAGG

[1]	Shu-Ping PENG, Cheng-Ming DONG, Yun-Hao ZHU. Cloning and Expression Analysis of Two Key Genes of Jasmonic Acid Synthesis in Response to Endophytic Infection from Rehmannia glutinosa [J]. Bulletin of Botanical Research, 2021, 41(2): 294-301.
[2]	WANG Wan-Qi, QI Wan-Zhu, ZHAO Qiu-Shuang, ZENG Dong, LIU Yi, FU Peng-Yue, QU Guan-Zheng, ZHAO Xi-Yang. Cloning and Expression Analysis of BpJMJ18 Gene Promoter in Betula platyphylla [J]. Bulletin of Botanical Research, 2020, 40(5): 751-759.
[3]	JIANG Cheng, ZHANG Xi, TIAN Qing, LI Li. Isolation of the BpbHLH112 Gene and Expression Analysis of Its Promoter in Betula platyphylla [J]. Bulletin of Botanical Research, 2020, 40(4): 583-592.
[4]	MA Miao-Miao, LI Cheng-Hao, LIU Xiao, ZHANG Xin, YANG Jing-Li. Cloning and Bioinformatics Analysis of LoERF017 Gene from Larix kaempferi [J]. Bulletin of Botanical Research, 2020, 40(4): 602-612.
[5]	ZHANG Yu-Qing, LIU Ye, QU Chun-Pu, LIU Guan-Jun, YANG Tian-Tian, YANG Cheng-Jun. Resistance Analysis of Transgenic PnDof30 Arabidopsis under Abiotic Stress [J]. Bulletin of Botanical Research, 2020, 40(3): 407-415.
[6]	LIU Jia-Li, HE Ming-Liang, LIU Chen-Xi, LIAO Xu, LI Xiu-Feng, GUAN Qing-Jie. Zinc Finger Protein OsZFP6 Expression Features and Functions in Saline-alkali Stress Response [J]. Bulletin of Botanical Research, 2020, 40(3): 424-432.
[7]	LI Ya-Bo, Lü Jia-Xin, TAN Bing, GAO Cai-Qiu. Cloning and expression analysis of 5 ZFP genes from Poplus trichocarpa [J]. Bulletin of Botanical Research, 2020, 40(2): 243-250.
[8]	LI Yang, SHI Ya-Kun, GAO Shi-Ke, Li Xiao-Yu, LAN Xing-Guo. Gene Cloning and Expression Analysis of PDIL1-2 of Ornamental Kale(Brassica oleracea var. acephala) [J]. Bulletin of Botanical Research, 2020, 40(2): 293-300.
[9]	QIN Lin-Lin, ZHANG Xi, JIANG Cheng, LI Li. Cloning and Functional Analysis of BpZFP4 Promoter from Birch(Betula platyphylla) [J]. Bulletin of Botanical Research, 2019, 39(6): 917-926.
[10]	LI Shuang, XIONG Ying, RALF M;ller-Xing, XING Qian. Distinct Expression Patterns of WRKY6 and PR1 in Arabidopsis Stress Memory Assays [J]. Bulletin of Botanical Research, 2019, 39(5): 752-759.
[11]	LI Xin-Yu, HE Li-Ming, ZHAN Ya-Guang. Cloning and Expression Analysis of FmWRKY44 Gene in Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2019, 39(4): 529-538.
[12]	BAI Xiao-Ming, DONG Shi-Wei, YANG Yu-Ning, SONG Yue, ZHANG Han-Guo, LI Shu-Juan. Cloning and Expression Analysis of Catalase Gene(LoCAT1) from Larix olgensis [J]. Bulletin of Botanical Research, 2019, 39(4): 539-546.
[13]	WANG Sheng-Yu, ZHANG Qi, ZHANG Zheng-Yi, HU Xiao-Qing, TIAN Jing, ZHANG Yong, LIU Xue-Mei. Cloning and Expression Analysis of BpSPL2 Promoter from Betula platyphylla [J]. Bulletin of Botanical Research, 2019, 39(1): 96-103.
[14]	DING Yi-Wei, ZHAN Ya-Guang, ZHANG Jia-Wei, HE Li-Ming. Cloning and Expression Analysis of Two PLT Transcription Factors Genes in Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2019, 39(1): 139-147.
[15]	WANG Heng-Tao, SHAO Wan-Xuan, XU Shu-Hao, ZENG Fan-Suo. Cloning and Expression Pattern Analysis of MUR5 Gene in Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2018, 38(6): 913-920.