Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’

doi:10.16420/j.issn.0513-353x.2022-1185

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (10): 2069-2078.doi: 10.16420/j.issn.0513-353x.2022-1185

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Next Articles

Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’

WU Xiulan¹, LI Guihua³, TANG Wenwu²^,^*()

¹ College of Food and Pharmaceutical Engineering，Zhaoqing University，Zhaoqing，Guangdong 526061，China
² College of Life Sciences，Zhaoqing University，Zhaoqing，Guangdong 526061，China
³ Vegetable Research Institute，Guangdong Academy of Agricultural Sciences，Guangzhou 510640，China

Received:2023-02-06 Revised:2023-08-25 Online:2023-10-25 Published:2023-10-30

Abstract

Abstract:

Ubiquitin-conjugating enzyme E2 is a key enzyme for substrate ubiquitination reaction，which plays an important role in the mechanism of self-incompatible by S-RNase degradation via ubiquitin-proteasome system. An E2 gene was cloned based on EST sequence（EY715921）of Citrus sinensis using the flower buds from‘Wuzi Shatangju’and‘Shatangju’，named as CrUBC2. The full-length cDNA sequence of the CrUBC2 was 965 bp，encoding 152 amino acids. There was one base difference （C/T）in the coding sequence of CrUBC2 between‘Wuzi Shatangju’and‘Shatangju’，which resulted in the conversion of threonine to isoleucine. Multiple sequence alignment showed that only one amino acid difference（Thr/Ile）was found between CrUBC2 and orthologs. Therefore，CrUBC2 protein was highly conservative during the evolution process. qRT-PCR showed that the highest expression level of the CrUBC2 was detected in anthers，and the expression level of the CrUBC2 after cross-pollination was 2.22-5.36 fold higher than that of self-pollination，indicating obvious expression specificity in different pollination. Pollen germination experiment in vitro showed that CrUBC2 protein of‘Wuzi Shatangju’inhibited significantly the growth of pollen tubes of self-pollination，but it had no effect on the growth of cross-pollination pollen tubes，which may be related to its involvement in S-RNase ubiquitination and resulting in cross-compatibility.

Key words: Citrus reticulata, CrUBC2, self-incompatible, gene cloning, function analysis

WU Xiulan, LI Guihua, TANG Wenwu. Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’[J]. Acta Horticulturae Sinica, 2023, 50(10): 2069-2078.

Figures/Tables 8

Table 1 Primers for experiment

引物名称Primer	引物序列（5′-3′）Primer sequence
CrUBC2-F1	TAGGCTCGACTCAACACACGCAA
CrUBC2-R1	CGAGCTCCACCTTTAATAGAGGTG
CrUBC2-F2	CATGTCGACACCTGCAAGGA
CrUBC2-R2	CCTTAATCTGCTGTCCAGCTC
qCrUBC2-F	GCAAGGAAGAGGCTGATGAGAGA
qCrUBC2-R	CAAATCGCACAGTTGGCGGTTTG
Actin-F	CCAATTCTCTCTTGAACCTGTCCTT
Actin-R	TGACTGATGAGAACTGCCAGAAG
GFPCrUBC2-F	CAAATTCGCGACCGGTATGTCGACACCTGCAAGGAAGAG
GFPCrUBC2-R	TGCTAGCCATACCGGTATCTGCTGTCCAGCTCTGTTCCA
pET32a-CrUBC2-F	CGGAATTCATGTCGACACCTGCAAG
pET32a-CrUBC2-R	CGCGTCGAATCTGCTGTCCAGCTCT
GFP-UBE2-F	CAAATTCGCGACCGGTATGTCGACACCTGCAAGGAAGAG
GFP-UBE2-R	TGCTAGCCATACCGGTATCTGCTGTCCAGCTCTGTTCCA

Fig. 1 Sequence alignment of CrUBC2 gene between‘Shatangju’and‘Wuzi Shatangju’

Fig. 2 Alignments of protein sequences of CrUBC2 and other UBC2

Fig. 3 Expression analyses of CrUBC2 gene in different tissues of‘Wuzi Shatangju’and‘Shatangju’ ** or * on the bars indicate significant differences between‘Wuzi Shatangju’and‘Shatangju’at 0.01 or 0.05 level.

Fig. 4 Expression analyses of CrUBC2 genes in different stages after self-pollination and cross-pollination Different lowercase letters on the bars indicate significant differences among different times（P < 0.05）. ** indicate significant differences between self-pollination and cross-pollination（P < 0.01）.

Fig. 5 Subcellular localization analysis of CrUBC2 in tobacco

Fig. 6 Heterologous expression protein of CrUBC2 using E. coli expression system in‘Wuzi Shatangju’（A）and‘Shatangju’（B）

Fig. 7 Effects of CrUBC2 protein from‘Wuzi Shatangju’on pollen tube length of‘Wuzi Shatangju’and‘Shatangju’ Different lowercase letters indicate significant differences between treatments with‘Wuzi Shatangju’（P < 0.05），** indicates significant differences between‘Wuzi Shatangju’and‘Shatangju’（P < 0.01）.

References 34

[1]	Chai L, Ge X, Biswas M K, Deng X. 2011a. Molecular analysis and expression of a floral organ-relative F-box gene isolated from‘Zigui Shatian’pummelo(Citrus grandis Osbeck). Molecular Biology Report, 38 (7):4429-4436.
[2]	Chai L, Ge X, Xu Q, Deng X. 2011b. CgSL2,an S-like RNase gene in‘Zigui Shatian’pummelo(Citrus grandis Osbeck),is involved in ovary senescence. Molecular Biology Report, 38 (1):1-8.
[3]	Deng Xiuxin. 2022. A review and perspective for citrus breeding in China during the last six decades. Acta Horticulturae Sinica, 49 (10):2063-2074. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0701 URL
	邓秀新. 2022. 中国柑橘育种60年回顾与展望. 园艺学报, 49 (10):2063-2074. doi: 10.16420/j.issn.0513-353x.2021-0701 URL
[4]	Entani T, Kubo K, Isogai S, Fukao Y, Shirakawa M, Isogai A, Takayama S. 2014. Ubiquitin-proteasome-mediated degradation of S-RNase in a solanaceous cross-compatibility reaction. Plant Journal, 78:1014-1021. doi: 10.1111/tpj.2014.78.issue-6 URL
[5]	Gil K E, Kim W Y, Lee H J, Faisal M, Saquib Q, Alatar A A, Park C M. 2017. ZEITLUPE contributes to a thermoresponsive protein quality control system in Arabidopsis. Plant Cell, 29 (11):2882-2894. doi: 10.1105/tpc.17.00612 URL
[6]	He M, Li L, Xu Y, Mu J, Xie Z, Gu C, Zhang S. 2022. Identification of S-genotypes and a novel S-RNase in 84 native Chinese pear accessions. Horticultural Plant Journal, 8 (6):1-14. doi: 10.1016/j.hpj.2021.04.010 URL
[7]	He Min, Gu Chao,Wu Juyou,Zhang Shaoling. 2021. Recent advances on self-incompatibility mechanism in fruit trees. Acta Horticulturae Sinica, 48 (4):759-777. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0425 URL
	何敏, 谷超, 吴巨友, 张绍铃. 2021. 果树自交不亲和机制研究进展. 园艺学报, 48 (4):759-777. doi: 10.16420/j.issn.0513-353x.2020-0425 URL
[8]	Hua Z H, Fields A, Kao T H. 2008. Biochemical models for S-RNase-based self-incompatibility. Molecular Plant, 1 (4):575-585. doi: 10.1093/mp/ssn032 URL
[9]	Isono E, Nagel M K. 2014. Deubiquitylating enzymes and their emerging role in plant biology. Front Plant Science, 5:56.
[10]	Li P, Miao H, Ma Y, Wang L, Hu G, Ye Z, Zhao J, Qin Y. 2015. CrWSKP1,an SKP1-like gene,is involved in the self-incompatibility reaction of“Wuzishatangju”(Citrus reticulata Blanco). International Journal Molecular Science, 16 (9):21695-21710. doi: 10.3390/ijms160921695 URL
[11]	Li Peng, Miao Hong-xia, Hu Gui-bing, Qin Yong-hua. 2019. Functional analyses of WUBE1 gene isolated from Citrus reticulata‘Wuzishatangju’. Journal of Tropical and Subtropical Botany, 27 (4):408-414. (in Chinese)
	李鹏, 苗红霞, 胡桂兵, 秦永华. 2019. 无籽沙糖桔WUBE1基因的功能验证. 热带亚热带植物学报, 27 (4):408-414.
[12]	Liang M, Cao Z, Zhu A, Liu Y, Tao M, Yang H, Xu Q Jr, Wang S, Liu J, Li Y, Chen C, Xie Z, Deng C, Ye J, Guo W, Xu Q, Xia R, Larkin R M, Deng X, Bosch M, Franklin-Tong V E, Chai L. 2020. Evolution of self-compatibility by a mutant Sm-RNase in Citrus. Nature Plants, 6 (2):131-142. doi: 10.1038/s41477-020-0597-3 pmid: 32055045
[13]	Liu W, Tang X, Qi X, Fu X, Ghimire S, Ma R, Li S, Zhang N, Si H. 2020. The ubiquitin conjugating enzyme:an important ubiquitin transfer platform in ubiquitin-proteasome system. International Journal Molecular Science, 21 (8):2894. doi: 10.3390/ijms21082894 URL
[14]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[15]	Miao H X, Qin Y H, Teixeira da Silva J A, Ye Z X, Hu G B. 2011. Cloning and expression analysis of S-RNase homologous gene in Citrus reticulata Blanco cv. Wuzishatangju. Plant Science, 180 (2):358-367. doi: 10.1016/j.plantsci.2010.10.012 URL
[16]	Miao H X, Qin Y H, Ye Z X, Hu G B. 2013. Molecular characterization and expression analysis of ubiquitin-activating enzyme E 1 gene in Citrus reticulata. Gene, 513 (2):249-259. doi: 10.1016/j.gene.2012.10.056 URL
[17]	Nagels D A, Pauwels L, Goossens A. 2016. The ubiquitin system and jasmonate signaling. Plants, 5 (1):6. doi: 10.3390/plants5010006 URL
[18]	Nie Wenfeng, Wang Jinyu, Gao Chunjuan, Chen Xuehao. 2022. A review on epigenetic modifications in regulating fruit development of horticultural crops. Acta Horticulturae Sinica, 49 (3):671-686. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0161 URL
	聂文锋, 王金玉, 高春娟, 陈学好. 2022. 表观遗传修饰调控园艺植物果实发育研究进展. 园艺学报, 49 (3):671-686. doi: 10.16420/j.issn.0513-353x.2021-0161 URL
[19]	Qiao H, Wang H, Zhao L, Zhou J, Huang J, Zhang Y, Xue Y. 2004. The F-box protein AhSLF-S 2 physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum. Plant Cell, 16 (3):582-595. doi: 10.1105/tpc.017673 URL
[20]	Ren Y, Hua Q, Pan J, Zhang Z, Zhao J, He X, Qin Y, Hu G. 2020. SKP1-like protein,CrSKP1-e,interacts with pollen-specific F-box proteins and assembles into SCF-type E3 complex in‘Wuzishatangju’(Citrus reticulata Blanco)pollen. Peer Journal, 8:e10578. doi: 10.7717/peerj.10578 URL
[21]	Sharma B, Joshi D, Yadav P K, Gupta A K, Bhatt T K. 2016. Role of ubiquitin-mediated degradation system in plant biology. Front Plant Science, 7:806.
[22]	Shu K, Yang W. 2017. E 3 ubiquitin ligases:ubiquitous actors in plant development and abiotic stress responses. Plant Cell Physiology, 58:1461-1476. doi: 10.1093/pcp/pcx071 URL
[23]	Stone S L, Anderson E M, Mullen R T, Goring D R. 2003. ARC 1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell, 15 (4):885-898. doi: 10.1105/tpc.009845 URL
[24]	Sun P, Li S, Lu D, Williams J S, Kao T H. 2015. Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation. Plant Journal, 83 (2):213-223. doi: 10.1111/tpj.2015.83.issue-2 URL
[25]	Tang W W, Wu X L. 2017. Molecular characterization and expression analysis of ring-box protein1(Rbx1)gene in Citrus reticulata. Research Journal of Biotechnology, 12:1-6.
[26]	Tang Wen-wu, Mo Can-kun, Wu Xiu-lan. 2018. Cloning and expression analysis of U-box gene,an E 3 ubiquitin ligase,in‘Wuzhishatangju’(Citrus reticulata Blanco). Acta Agriculturae Universitatis Jiangxiensis, 40 (2):383-388. (in Chinese)
	唐文武, 莫灿坤, 吴秀兰. 2018. 无籽沙糖桔E3泛素连接酶U-box基因的克隆及表达分析. 江西农业大学学报, 40 (2):383-388.
[27]	Vierstra R. 2009. The ubiquitin-26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10:385-397. doi: 10.1038/nrm2688 pmid: 19424292
[28]	Wang F, Deng X. 2011. Plant ubiquitin-proteasome pathway and its role in gibberellin signaling. Cell Research, 21:1286-1294. doi: 10.1038/cr.2011.118 pmid: 21788985
[29]	Wang Lirong, Wu Jinlong. 2021. Review for the research of fruit tree germplasm and breeding of new varieties in the past seven decades in China. Acta Horticulturae Sinica, 48 (4):749-758. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0616 URL
	王力荣, 吴金龙. 2021. 中国果树种质资源研究与新品种选育70年. 园艺学报, 48 (4):749-758. doi: 10.16420/j.issn.0513-353x.2020-0616 URL
[30]	Wei Zhuangmin, Wei Sijia, Chen Peng, Hu Jianbing, Tang Yuqing, Ye Junli, Li Xianxin, Deng Xiuxin, Chai Lijun. 2022. Identification of S-genotypes of 63 pummelo germplasm resources. Acta Horticulturae Sinica, 49 (5):1111-1120. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0260
	韦壮敏, 魏斯佳, 陈鹏, 胡健兵, 汤雨晴, 叶俊丽, 李先信, 邓秀新, 柴利军. 2022. 63份柚类资源S基因型鉴定. 园艺学报, 49 (5):1111-1120. doi: 10.16420/j.issn.0513-353x.2021-0260
[31]	Wu X L, Tang W W. 2016. Cloning and expression analysis of the Cullin gene in Citrus reticulata. Research Journal of Biotechnology, 11 (1):109-114.
[32]	Yu Qinhan, Jiao Shuzhen, Wu Nan, Zhang Ningbo, Xu Weirong. 2021. Molecular cloning,expression and polyclonal antibody preparation of E 3 ubiquitin ligase gene HOS1 from Vitis vinifera. Acta Horticulturae Sinica, 48 (6):1173-1182. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0557 URL
	俞沁含, 焦淑珍, 吴楠, 张宁波, 徐伟荣. 2021. 葡萄E3泛素酶HOS1基因克隆、表达及抗血清制备. 园艺学报, 48 (6):1173-1182. doi: 10.16420/j.issn.0513-353x.2020-0557 URL
[33]	Zhao H, Song Y, Li J, Zhang Y, Huang H, Li Q, Zhang Y, Xue Y. 2021. Primary restriction of S-RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida. New Phytologist, 231 (3):1249-1264. doi: 10.1111/nph.v231.3 URL
[34]	Zwirn P, Stary S, Luschnig C, Bachmair A. 1997. Arabidopsis thaliana RAD6 homolog AtUBC2 complements UV sensitivity,but not N-end rule degradation deficiency,of Saccharomyces cerevisiae rad6 mutants. Current Genetics, 32 (5):309-314. pmid: 9371881

Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 8

References 34

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	GAO Pengfei, GAO Bing, FENG Zhenghong, WU Jianhui. Cloning and Cd-resistant Analysis of PsWRKY40 in Potentilla sericea [J]. Acta Horticulturae Sinica, 2023, 50(6): 1269-1283.
[2]	YIN Xiaoyu, HE Guoren, BI Mengmeng, TANG Yuchao, HAO Chunlian, QU Yuxiao, HAO Zehui, XU Leifeng, HU Fengrong, YANG Panpan, MING Jun. Cloning and Functional Analysis of LlLBD18 in Lilium lancifolium [J]. Acta Horticulturae Sinica, 2023, 50(10): 2117-2127.
[3]	WANG Sha, ZHANG Xinhui, ZHAO Yujie, LI Bianbian, ZHAO Xueqing, SHEN Yu, DONG Jianmei, YUAN Zhaohe. Cloning and Functional Analysis of PgMYB111 Related to Anthocyanin Synthesis in Pomegranate [J]. Acta Horticulturae Sinica, 2022, 49(9): 1883-1894.
[4]	QIU Ziwen, LIU Linmin, LIN Yongsheng, LIN Xiaojie, LI Yongyu, WU Shaohua, YANG Chao. Cloning and Functional Analysis of the MbEGS Gene from Melaleuca bracteata [J]. Acta Horticulturae Sinica, 2022, 49(8): 1747-1760.
[5]	ZHENG Lin, WANG Shuai, LIU Yunuo, DU Meixia, PENG Aihong, HE Yongrui, CHEN Shanchun, ZOU Xiuping. Gene Cloning and Expression Analysis of NAC Gene in Citrus in Response to Huanglongbing [J]. Acta Horticulturae Sinica, 2022, 49(7): 1441-1457.
[6]	LIU Yaoyao, WU Yanyan, Shi Yan, MAO Tianyu, BAO Manzhu, ZHANG Junwei, ZHANG Jie. Preliminary Study on the Relationship Between Promoter Sequence Difference of PmTAC1 and Weeping Trait of Prunus mume [J]. Acta Horticulturae Sinica, 2022, 49(6): 1327-1338.
[7]	XIANG Li, ZHAO Lei, WANG Mei, LÜ Yi, WANG Yanfang, SHEN Xiang, CHEN Xuesen, YIN Chengmiao, MAO Zhiquan. Cloning and Functional Analysis of MdWRKY74 in Apple [J]. Acta Horticulturae Sinica, 2022, 49(3): 482-492.
[8]	SONG Mengfei, ZHA Gaohui, CHEN Jinfeng, LOU Qunfeng. Research Progress on Molecular Basis of Plant Architecture Related Traits in Cucumber [J]. Acta Horticulturae Sinica, 2022, 49(12): 2683-2702.
[9]	SUN Wei, SUN Shiyu, CHEN Yiran, WANG Yuhan, ZHANG Yan, JU Zhigang, YI Yin. Cloning and Function Analysis of Chalcone Isomerase Gene RdCHI1 in Rhododendron delavayi [J]. Acta Horticulturae Sinica, 2022, 49(11): 2407-2418.
[10]	ZENG Yike, SHI Ying, CHEN Siyi, LI Guojing, HUANG Xianbiao, XIE Zongzhou, LI Chunlong, GUO Dayong, LIU Jihong. Effects of Film Mulching on Improving Fruit Quality of Ponkan and Possible Mechanisms [J]. Acta Horticulturae Sinica, 2022, 49(11): 2419-2430.
[11]	LI Maofu, YANG Yuan, WANG Hua, FAN Youwei, SUN Pei, JIN Wanmei. Identification and Analysis of Self Incompatibility S-RNase in Rose [J]. Acta Horticulturae Sinica, 2022, 49(1): 157-165.
[12]	XU Ligong, HAN Taili, SUN Jifeng, YANG Xiaodong, TAN Jinxia, SONG Yinhang, LI Meng, ZHOU Luhong, and SUN Shasha. A New Spring Chinese Cabbage Cultivar‘Weichun 22’ [J]. Acta Horticulturae Sinica, 2021, 48(S2): 2825-2826.
[13]	BIAN Shicun, LU Yani, XU Wujun, CHEN Boqing, WANG Guanglong, XIONG Aisheng. Garlic Circadian Clock Genes AsRVE1 and AsRVE2 and Their Expression Analysis Under Osmotic Stress [J]. Acta Horticulturae Sinica, 2021, 48(9): 1706-1716.
[14]	MA Junjie, SONG Lina, LI Le, MA Xiaochun, JIN Lei, XU Weirong. VaCBL6 from Vitis amurensis Involved in Abiotic Stress Response and ABA-mediated Pathway [J]. Acta Horticulturae Sinica, 2021, 48(6): 1079-1093.
[15]	YU Hailong, REN Wenjing, FANG Zhiyuan, YANG Limei, ZHUANG Mu, LÜ Honghao, WANG Yong, JI Jialei, ZHANG Yangyong. Advances on Fertility Restoration for Cytoplasmic Male Sterility in Vegetables [J]. Acta Horticulturae Sinica, 2021, 48(5): 1031-1046.