葡萄E3泛素酶HOS1基因克隆、表达及抗血清制备

doi:10.16420/j.issn.0513-353x.2020-0557

摘要/Abstract

摘要：

HOS1（High expression of osmotically responsive genes 1）编码1个具有E3泛素连接酶活性的蛋白,是模式植物拟南芥冷反应信号转导途径的关键负调控因子,但其在多年生葡萄属植物中参与低温胁迫应答的分子机制尚不明确。以欧洲酿酒葡萄品种‘霞多丽'的叶片为供试材料,克隆了VvHOS1基因,其全长2 931 bp,编码976个氨基酸,在第74 ~ 106个氨基酸处存在RING类型的锌指结构域（zf-C3H4_3）。系统发育分析表明,葡萄属植物的HOS1蛋白与番茄、烟草、茶树的亲缘关系最近。将VvHOS1抗原表位丰富肽段（1 ~ 320 aa）构建至原核表达载体pET28a-SUMO的BamHⅠ与Hind Ⅲ位点,转化大肠杆菌。经IPTG诱导发现,pET28a- VvHOS1^1-320在大肠杆菌Rosetta（DE3）中高表达,融合蛋白主要以包涵体形式存在。将纯化后的重组蛋白作为抗原免疫大耳白兔,获得anti-VvHOS1多克隆抗体,ELISA检测效价为1︰51 200,具有较强的Western特异性。该抗血清能够特异性检测‘霞多丽'葡萄叶片中的VvHOS1蛋白。（4 ℃）低温胁迫6、36与48 h时VvHOS1上调表达显著。

关键词: 葡萄, HOS1, 原核表达, 多克隆抗体, 低温胁迫, 表达模式分析

Abstract:

HOS1（High expression of osmotically responsive genes 1）encodes a protein with E3 ubiquitin ligase activity,and is a key negative regulator of cold response signal transduction pathway of model plant Arabidopsis,but its molecular mechanisms involved in the low temperature stress response in perennial Vitis vinifera are unclear. The VvHOS1 gene was cloned from the leaves of Vitis vinifera ‘Chardonnay'. The full length of VvHOS1 was 2 931 bp,coding 976 aa,and the presence of a RING-type zinc finger domain was located at 74-106 aa（zf-C3H4_3）. Phylogenetic tree analysis showed that VvHOS1 protein in Vitis vinifera is most closely related to tomato,tobacco,and tea tree. Furthermore, the VvHOS1 antigen epitope rich fragment（1-320 aa）was constructed into the prokaryotic expression vector pET28a-SUMO at the BamHⅠandHind Ⅲ restriction sites. The pET28a- VvHOS1^1-320was found to be highly expressed inEscherichia coliRosetta（DE3）with isopropyl-β-D-thiogalactoside（IPTG）induction. The fusion proteins were mainly in the form of inclusion body proteins. The purified recombinant protein was used as antigen to immunize white rabbits,and the resulting anti-VvHOS1 polyclonal antibody was obtained and detected by ELISA. The potency was 1︰51 200 with a strong Western specificity. The antiserum specifically detects VvHOS1 protein in grapevine leaves after cold stress（4 ℃）,and Western blot analysis indicated that the expression of VvHOS1 was significantly up-regulated at 6,36 and 48 h.

Key words: grape, HOS1, prokaryotic expression, polyclonal antibody, cold stress, expression pattern analysis

中图分类号:

S663.1

俞沁含, 焦淑珍, 吴楠, 张宁波, 徐伟荣. 葡萄E3泛素酶HOS1基因克隆、表达及抗血清制备[J]. 园艺学报, 2021, 48(6): 1173-1180.

YU Qinhan, JIAO Shuzhen, WU Nan, ZHANG Ningbo, XU Weirong. Molecular Cloning,Expression and Polyclonal Antibody Preparation of E3 Ubiquitin Ligase Gene HOS1 from Vitis vinifera[J]. Acta Horticulturae Sinica, 2021, 48(6): 1173-1180.

图/表 9

图1 VvHOS1基因的RT-PCR扩增产物

Fig. 1 RT-PCR products of VvHOS1 gene

图2 VvHOS1蛋白结构域预测 Zf-C3H4_3：C3H4类型的RING finger结构域;ELYS：原子卵黄囊的胚胎大分子;直线：用于原核表达的区段。

Fig. 2 Prediction of VvHOS1 protein domain Zf-C3H4_3：RING finger domain of C3H4 type;ELYS：Embryonic large molecule derived from yolk sac;Line：Region used for prokaryotic expression.

图3 VvHOS1进化分析

Fig. 3 Phylogenetic analysis of VvHOS1

图4 pET28a-VvHOS11-320的原核表达分析 A：pET28a-VvHOS11-320重组原核表达载体的构建流程;B：VvHOS11-32050 kD蛋白诱导表达。 M：蛋白质分子量标准;CK：pET28a空载体诱导表达。

Fig. 4 Analysis of prokaryotic expression of pET28a-VvHOS11-320 A：The construction process of fusion protein prokaryotic vector pET28a-VvHOS11-320;B：Prokaryotic protein induced expression of pET28a-VvHOS11-320. M：Protein molecular weight standard;CK：Induced expression products of pET28a empty vector.

图5 重组蛋白的可溶性分析 1：0.4 mg · mL -1 BSA;M：蛋白质分子量标准;2：上清液;3：上清液（2 mol · L -1尿素溶解包涵体）;4、5：包涵体2倍和10倍稀释（8 mol · L -1尿素溶解包涵体）;6：pET28a-SUMO空载体诱导表达。

Fig. 5 Assay of solubility of recombinant protein 1：0.4 mg ∙ mL -1 BSA;M：Protein molecular weight standard;2：Lysis supernatant;3：The supernatant（dissolved by 2 mol · L -1urea）;4,5：Inclusion bodies of 2-fold and 10-fold diluents （dissolved by 8 mol · L -1 urea）;6：Induced expression of empty vector pET28a.

图6 VvHOS11-320 50 kD融合蛋白纯化的SDS-PAGE 1、2：0.4 mg · mL -1 BSA;M：蛋白质分子量标准;3、4：包涵体10倍和20倍稀释（6 mol · L -1盐酸胍溶解包涵体）。

Fig. 6 SDS-PAGE analysis of VvHOS11-320 50 kD purified fusion protein 1,2：0.4 mg ∙ mL -1 BSA;M：Protein molecular weight standard;3,4：10-fold and 20-fold dilution of inclusions （6 mol · L -1 guanidine hydrochloride dissolving inclusion bodies）.

图7 VvHOS1多克隆抗体效价分析

Fig. 7 Analysis of anti-VvHOS1 titer by ELISA

图8 制备的多克隆抗体Western blot分析

Fig. 8 Western blot analysis of the antiserum

图9 Western blot 检测欧洲葡萄HOS1蛋白 4 ℃低温胁迫后‘霞多丽'叶片总蛋白的Western blot结果。

Fig. 9 Western blot analysis Vitis vinifera‘Chardonnay'HOS1 protein Western blot of the leave total proteins treated with low temperature（4 ℃）.

参考文献 31

[1]	An J P, Liu X, Song L Q, You C X, Wang X F, Hao Y J. 2017. Functional characterization of the apple RING E3 ligase MdMIEL1 in transgenicArabidopsis. Horticultural Plant Journal, 3 (2):53-59. doi: 10.1016/j.hpj.2017.01.001 URL
[2]	Chen Xiu, Rao Xueqin, Ruan Xiaolei, Liu Fuxiu, Li Huaping. 2013. Prokaryotic expression and antiserum preparation of functional domain of banana streak virus coat protein gene. Acta Horticulturae Sinica, 40 (12):2401-2408. (in Chinese)
	陈秀, 饶雪琴, 阮小蕾, 刘福秀, 李华平. 2013. 香蕉线条病毒衣壳蛋白功能域基因的原核表达及抗血清制备. 园艺学报, 40 (12):2401-2408.
[3]	Cheng L, Zhang W, Hu J, Zhang Z, Liu Y. 2020a. Characterization of the key region and key phosphorylation sites of EcaICE 1 for its molecular interaction with EcaHOS1 protein in Eucalyptus camaldulensis. Plant Biology, 23 (2):400-406. doi: 10.1111/plb.v23.2 URL
[4]	Cheng Z, Zhang X, Huang P, Huang G, Wang X. 2020b. Nup96 and HOS 1 are mutually stabilized and gate CONSTANS protein level,conferring long-day photoperiodic flowering regulation in Arabidopsis. The Plant Cell, 32 (2):374-391. doi: 10.1105/tpc.19.00661 URL
[5]	Ding Y, Li H, Zhang X, Xie Q, Gong Z, Yang S. 2015. OST 1 Kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis. Developmental Cell, 32 (3):278-289. doi: 10.1016/j.devcel.2014.12.023 URL
[6]	Dong C H, Agarwal M, Zhang Y, Xie Q, Zhu J K. 2006. The negative regulator of plant cold responses,HOS1,is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proceedings of the National Academy of Science, 103 (21):8281-8286.
[7]	Gillenius P, Jäätmaa E, Askelöf P, Granström M, Tiru M. 1985. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. Journal of Biological Standardization, 13 (1):61-66. pmid: 4039324
[8]	Hershko A, Heller H, Elias S, Ciechanover A. 1983. Components of ubiquitin-protein ligase system. Resolution,affinity purification,and role in protein breakdown. Journal of Biological Chemistry, 258 (13):8206-8214. doi: 10.1016/S0021-9258(20)82050-X URL
[9]	Ishitani M, Xiong L, Lee H, Stevenson B, Zhu J K. 1998. HOS1,a genetic locus involved in cold-responsive gene expression in Arabidopsis. The Plant Cell, 10 (7):1151-1161. doi: 10.1105/tpc.10.7.1151 URL
[10]	Jung J-H, Park J-H, Lee S, To T K, Kim J M, Seki M, Park C M. 2013. The cold signaling attenuator HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 activates FLOWERING LOCUS C transcription via chromatin remodeling under short-term cold stress in Arabidopsis. The Plant Cell, 25 (11):4378-4390. doi: 10.1105/tpc.113.118364 URL
[11]	Kim J H, Lee H J, Park C M. 2017. HOS 1 acts as a key modulator of hypocotyl photomorphogenesis. Plant Signaling & Behavior, 12 (5):e1315497.
[12]	Kurepa J, Toh-e A, Smalle J A. 2008. 26S proteasome regulatory particle mutants have increased oxidative stress tolerance. The Plant Journal, 53 (1):102-114. doi: 10.1111/tpj.2008.53.issue-1 URL
[13]	Lazaro A, Mouriz A, Piñeiro M, Jarillo J A. 2015. Red light-mediated degradation of CONSTANS by the E 3 ubiquitin ligase HOS1 regulates photoperiodic flowering inArabidopsis. The Plant Cell, 27 (9):2437-2454. doi: 10.1105/tpc.15.00529 pmid: 26373454
[14]	Lazaro A, Valverde F, Piñeiro M, Jarillo J A. 2012. The Arabidopsis E 3 ubiquitin ligase HOS1 negatively regulates CONSTANS abundance in the photoperiodic control of flowering. The Plant Cell, 24 (3):982-999. doi: 10.1105/tpc.110.081885 pmid: 22408073
[15]	Lee H, Xiong L, Gong Z, Ishitani M, Stevenson B, Zhu J K. 2001. The Arabidopsis HOS 1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning. Genes & Development, 15 (7):912-924. doi: 10.1101/gad.866801 URL
[16]	Lee J H, Kim J J, Kim S H, Cho H J, Kim J, Ahn J H. 2012. The E 3 ubiquitin ligase HOS1 regulates low ambient temperature-responsive flowering in Arabidopsis thaliana. Plant and Cell Physiology, 53 (10):1802-1814. doi: 10.1093/pcp/pcs123 URL
[17]	Lee K, Seo P J. 2015. The Arabidopsis E 3 ubiquitin ligase HOS1 contributes to auxin biosynthesis in the control of hypocotyl elongation. Plant Growth Regulation, 76 (2):157-165. doi: 10.1007/s10725-014-9985-x URL
[18]	Li C, Wang J, Ji N, Lei C, Zhou D, Zheng Y, Wang K. 2020. PpHOS1,a RING E 3 ubiquitin ligase,interacts with PpWRKY22 in the BABA-induced priming defense of peach fruit against Rhizopus stolonifer. Postharvest Biology Technology, 159:111029. doi: 10.1016/j.postharvbio.2019.111029 URL
[19]	Li H, Jiang H, Bu Q, Zhao Q, Sun J, Xie Q, Li C. 2011. The Arabidopsis RING finger E 3 ligase RHA2b acts additively with RHA2a in regulating abscisic acid signaling and drought response. Plant Physiology, 156 (2):550-563. doi: 10.1104/pp.111.176214 URL
[20]	Li J, Wang N, Wang L, Xin H, Li S. 2014. Molecular cloning and characterization of the HOS1 gene from‘Muscat Hamburg'grapevine. Journal of the American Society for Horticultural Science, 139 (1):54-62. doi: 10.21273/JASHS.139.1.54 URL
[21]	Liu K, Wang L, Xu Y, Chen N, Ma Q, Li F, Chong K. 2007. Overexpression of OsCOIN,a putative cold inducible zinc finger protein,increased tolerance to chilling,salt and drought,and enhanced proline level in rice. Planta, 226 (4):1007-1016. doi: 10.1007/s00425-007-0548-5 URL
[22]	Lourenço T, Sapeta H, Figueiredo D D, Rodrigues M, Cordeiro A, Abreu I A, Saibo N J, Oliveira M M. 2013. Isolation and characterization of rice(Oryza sativa L.)E3-ubiquitin ligase OsHOS1 gene in the modulation of cold stress response. Plant Molecular Biology, 83 (4- 5):351-363.
[23]	Miura K, Ohta M, Nakazawa M, Ono M, Hasegawa P M J T P J. 2011. ICE 1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance. Plant Journal, 67 (2):269-279. doi: 10.1111/tpj.2011.67.issue-2 URL
[24]	Nandi D, Tahiliani P, Kumar A, Chandu D. 2006. The ubiquitin-proteasome system. Journal of Biosciences, 31 (1):137-155. doi: 10.1007/BF02705243 URL
[25]	Sullivan J A, Shirasu K, Deng X W. 2003. The diverse roles of ubiquitin and the 26S proteasome in the life of plants. Nature Reviews Genetics, 4 (12):948-958. pmid: 14631355
[26]	Vierstra R D. 2009. The ubiquitin-26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10 (6):385-397. doi: 10.1038/nrm2688 URL
[27]	Wang J Y, Liu Z J, Zhang G Q, Niu S C, Zhang Y Q, Peng C C. 2020. Evolution of two ubiquitin-like system of autophagy in orchid. Horticultural Plant Journal, 6 (5):321-334. doi: 10.1016/j.hpj.2020.05.006 URL
[28]	Xiao Yan, Lin Hua, Yang Lijuan, Chen Shijie, Xiong Zeping, Chen Qibing. 2013. Prokaryotic expression of Qiongzhuea tumidinoda CBF1 gene and preparation of its polyclonal antibody. Acta Horticulturae Sinica, 41 (2):375-381. (in Chinese)
	肖艳, 林华, 杨丽娟, 陈世界, 熊泽平, 陈其兵. 2013. 筇竹 CBF1基因的原核表达和多克隆抗体的制备. 园艺学报, 41 (2):375-381.
[29]	Xie Q, Guo H-S, Dallman G, Fang S, Weissman A M, Chua N-H. 2002. SINAT 5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals. Nature, 419 (6903):167-170.
[30]	Yan Shuangshuang, Qiu Zhengkun, Yu Bingwei, Ming Fangyan, Chen Changming, Lei Jianjun, Cao Bihao. 2020. Advances in phytohormone auxin response to high temperature. Acta Horticulturae Sinica, 47 (11):2238-2246. (in Chinese)
	颜爽爽, 邱正坤, 余炳伟, 明方艳, 陈长明, 雷建军, 曹必好. 2020. 植物生长素响应高温胁迫研究进展. 园艺学报, 47 (11):2238-2246.
[31]	Zhang Y, Yang C, Li Y, Zheng N, Chen H, Zhao Q, Gao T, Guo H, Xie Q. 2007. SDIR1 is a RING finger E 3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. The Plant Cell, 19 (6):1912-1929. doi: 10.1105/tpc.106.048488 URL