水稻组氨酸磷酸转运蛋白OsAHP2的表达及纯化

doi:10.7668/hbnxb.20190836

摘要/Abstract

摘要： 细胞分裂素在植物生长发育和抵御环境胁迫过程中均扮演着重要的角色，其信号是由细胞分裂素受体组氨酸激酶、组氨酸磷酸转运蛋白（HPs）以及反应调节因子组成的复杂的双元组分系统进行传递的。为了获得高纯度的OsAHP2蛋白，从水稻中扩增了OsAHP2全长cDNA，通过酶切连接的方法构建了2个OsAHP2原核表达载体，命名为pET-32a-OsAHP2和pGEX-4T-1-OsAHP2，测序正确后分别转化大肠杆菌表达菌株Rosseta和XA90。SDS-PAGE检测结果显示，0.5 mmol/L IPTG诱导1，3，5 h条件下，pET-32a-OsAHP2重组菌株表达出分子量约为35 ku的融合蛋白，pGEX-4T-1-OsAHP2重组菌株表达出分子量约为42 ku的融合蛋白，筛选出融合蛋白表达量较高的pET-32a-OsAHP2进行后续试验。在0.5 mmol/L IPTG诱导3 h条件下，重组大肠杆菌pET-32a-OsAHP2以可溶性蛋白的形式表达OsAHP2融合蛋白，通过His镍离子亲和层析柱纯化后，获得了大量纯度较好的OsAHP2融合蛋白。

关键词: 水稻, 组氨酸磷酸转运蛋白, OsAHP2, 原核表达, 蛋白纯化

Abstract: Cytokinin plays an important role in plant development and resistance to environmental stresses. Cytokinin signal transduction involves a multistep two-component system composed of a sensor histidine kinase (HK), a histidine phosphotransfer protein (HP), and a response regulator (RR). In order to obtain high purity OsAHP2 protein, the full-length cDNA of OsAHP2 was amplified from rice, and two prokaryotic expression vectors pET-32a-OsAHP2 and pGEX-4T-1-OsAHP2 were constructed. After sequencing, they were transformed into E. coli expression strains Rosseta and XA90,respectively. SDS-PAGE analysis showed that with 0.5 mmol/L IPTG induction for 1, 3, 5 h, the recombinant strain pET-32a-OsAHP2 expressed a 35 ku fusion protein and the recombinant strain pGEX-4T-1-OsAHP2 expressed a 42 ku fusion protein. The pET-32a-OsAHP2 expressed much more fusion protein than the pGEX-4T-1-OsAHP2 and was used for subsequent experiment. The pET-32a-OsAHP2 expressed fusion protein in the form of soluble protein by 0.5 mmol/L IPTG induction for 3 h. After purification by HIS-Select Nickel Affinity Gel, a large amount of OsAHP2 fusion protein with good purity was obtained.

Key words: Rice, Histidine phosphotransfer proteins, OsAHP2, Prokaryotic expression, Protein purification

中图分类号:

Q78
S511.03

孙丽静, 李倩影, 王培楠, 孙颖. 水稻组氨酸磷酸转运蛋白OsAHP2的表达及纯化[J]. 华北农学报, 2020, 35(4): 46-51. doi: 10.7668/hbnxb.20190836.

SUN Lijing, LI Qianying, WANG Peinan, SUN Ying. Protein Expression and Purification of OsAHP2 in Rice[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(4): 46-51. doi: 10.7668/hbnxb.20190836.

http://www.hbnxb.net/CN/Y2020/V35/I4/46

参考文献

[1] Schaller G E, Bishopp A, Kieber J J. The yin-yang of hormones:cytokinin and auxin interactions in plant development[J]. The Plant Cell, 2015, 27(1):44-63. doi:10.1105/tpc.114.133595.
[2] Brandizzi F. Divide, expand, differentiate-new insights on plant organ growth through cytokinin signaling[J]. The Plant Journal, 2019, 97(5):803-804. doi:10.1111/tpj.14261.
[3] Aki S S, Mikami T, Naramoto S, Nishihama R, Ishizaki K, Kojima M, Takebayashi Y, Sakakibara H, Kyozuka J, Kohchi T, Umeda M. Cytokinin signaling is essential for organ formation in Marchantia polymorpha[J]. Plant & Cell Physiology, 2019, 60(8):1842-1854. doi:10.1093/pcp/pcz100.
[4] Kieber J J, Schaller G E. Cytokinin signaling in plant development[J]. Development, 2018, 145(4):149344. doi:10.1242/dev.149344.
[5] Wang L, Chong K. The essential role of cytokinin signaling in root apical meristem formation during somatic embryogenesis[J]. Frontiers in Plant Science, 2016, 6:1196. doi:10.3389/fpls.2015.01196.
[6] Wybouw B, De Rybel B. Cytokinin-A developing story[J]. Trends in Plant Sience, 2019, 24(2):177-185. doi:10.1016/j.tplants.2018.10.012.
[7] Bielach A, Hrtyan M, Tognetti V B. Plants under stress:involvement of auxin and cytokinin[J]. International Journal of Molecular Sciences, 2017, 18(7):1427. doi:10.3390/ijms18071427.
[8] Huang X Z, Hou L Y, Meng J J, You H W, Li Z, Gong Z Z, Yang S H, Shi Y T. The antagonistic action of abscisic acid and cytokinin signaling mediates drought stress response in Arabidopsis[J]. Molecular Plant, 2018, 11(7):970-982. doi:10.1016/j.molp.2018.05.001.
[9] Li W Q, Herrera-Estrella L, Tran L P. Do cytokinins and strigolactones crosstalk during drought adaptation?[J]. Trends in Plant Science, 2019, 24(8):669-672. doi:10.1016/j.tplants.2019.06.007.
[10] Wang W C, Lin T C, Kieber J, Tsai Y C. Response regulator 9 and 10 negatively regulate salinity tolerance in rice[J]. Plant & Cell Physiology, 2019, 60(11):2549-2563.doi:10.1093/pcp/pcz149.
[11] Cortleven A, Leuendorf J E, Frank M, Pezzetta D, Bolt S, Schmülling T. Cytokinin action in response to abiotic and biotic stresses in plants[J]. Plant, Cell & Environment, 2019, 42(3):998-1018. doi:10.1111/pce.13494.
[12] Pavlů J, Novák J, Koukalová V, Luklová M, Brzobohatý B, Černý M. Cytokinin at the crossroads of abiotic stress signalling pathways[J]. International Journal of Molecular Sciences, 2018, 19(8):2450. doi:10.3390/ijms19082450.
[13] Heyl A, Schmülling T. Cytokinin signal perception and transduction[J]. Current Opinion in Plant Biology, 2003, 6(5):480-488. doi:10.1016/S1369-5266(03)00087-6.
[14] To J P C, Kieber J J. Cytokinin signaling:two-components and more[J]. Trends in Plant Science, 2008, 13(2):85-92. doi:10.1016/j.tplants.2007.11.005.
[15] Zürcher E, Müller B. Cytokinin synthesis, signaling, and function-advances and new insights[J]. International Review of Cell and Molecular Biology, 2016, 324:1-38. doi:10.1016/bs.ircmb.2016.01.001.
[16] Hutchison C E, Li J, Argueso C, Gonzalez M, Lee E, Lewis M W, Maxwell B B, Perdue T D, Schaller G E, Alonso J M, Ecker J R, Kieber J J. The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling[J]. The Plant Cell, 2006, 18(11):3073-3087. doi:10.1105/tpc.106.045674.
[17] Nishiyama R, Watanabe Y, Leyva-Gonzalez M A, Van Ha C, Fujita Y, Tanaka M, Seki M, Yamaguchi-Shinozaki K, Shinozaki K, Herrera-Estrella L, Phan Tran L S. Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response[J]. Proceedings of the National Academy of Sciences, 2013, 110(12):4840-4845. doi:10.1073/pnas.1302265110.
[18] Du L M, Jiao F C, Chu J, Jin G L, Chen M, Wu P. The two-component signal system in rice (Oryza sativa L.):a genome-wide study of cytokinin signal perception and transduction[J]. Genomics, 2007, 89(6):697-707. doi:10.1016/j.ygeno.2007.02.001.
[19] Schaller G E, Doi K, Hwang I, Kieber J J, Khurana J P, Kurata N, Mizuno T, Pareek A, Shiu S H, Wu P, Yip W K. Nomenclature for two-component signaling elements of rice[J]. Plant Physiology, 2007, 143(2):555-557. doi:10.1104/pp.106.093666.
[20] Tsai Y C, Weir N R, Hill K, Zhang W J, Kim H J, Shiu S H, Schaller G E, Kieber J J. Characterization of genes involved in cytokinin signaling and metabolism from rice[J]. Plant Physiology, 2012, 158(4):1666-1684. doi:10.1104/pp.111.192765.
[21] Sun L J, Zhang Q, Wu J X, Zhang L Q, Jiao W W, Zhang S W, Zhang Z G, Sun D Y, Lu T G, Sun Y. Two rice authentic histidine phosphotransfer proteins, OsAHP1 and OsAHP2, mediate cytokinin signaling and stress responses in rice[J]. Plant Physiology, 2014, 165(1):335-345. doi:10.1104/pp.113.232629.
[22] 苏文强, 胡鑫宇, 王秋霞, 欧长波, 郭爱疆, 李辉, 骆学农, 余燕, 张艳红, 姜金庆, 刘兴友, 马金友, 王松, 才学鹏. 猪囊尾蚴API基因原核表达条件的优化[J]. 西北农林科技大学学报(自然科学版), 2017, 45(8):1-6. doi:10.13207/j.cnki.jnwafu.2017.08.001. Su W Q, Hu X Y, Wang Q X, Ou C B, Guo A J, Li H, Luo X N, Yu Y, Zhang Y H, Jiang J Q, Liu X Y, Ma J Y, Wang S, Cai X P. Optimization of prokaryotic expression conditions for apoptosis protease inhibitor gene from Cysticercus cellulosae[J]. Journal of Northwest A & F University(Natural Science Edition), 2017, 45(8):1-6.
[23] 毛明光, 温施慧, 姜志强, 蒋洁兰, 孙航, 吕绘倩, 李幸. 太平洋鳕神经坏死病毒衣壳蛋白(CP)的原核表达及条件优化[J]. 大连海洋大学学报, 2016, 31(2):117-123. doi:10.16535/j.cnki.dlhyxb.2016.02.001. Mao M G, Wen S H, Jiang Z Q, Jiang J L, Sun H, Lü H Q, Li X. Prokaryotic expression and condition optimization of Nervous necrosis virus capsid protein(CP) in pacific cod Gadus macrocephalus[J]. Journal of Dalian Ocean University, 2016, 31(2):117-123.
[24] 周泽军, 庞欢瑛, 丁燏, 简纪常, 吴灶和. 溶藻弧菌HY9901转运蛋白TolB的原核表达及条件优化和纯化[J]. 中国农学通报, 2013, 29(11):55-59. doi:10.11924/j.issn.1000-6850.2012-3302. Zhou Z J, Pang H Y, Ding Y, Jian J C, Wu Z H. Purification and optimization of prokaryotic expression of translocation protein TolB gene from Vibrio alginolyticus strain HY9901[J]. Chinese Agricultural Science Bulletin, 2013, 29(11):55-59.
[25] 郝英辰, 龙月, 郭豪, 李宁, 张松杰, 赵棋, 张松涛. 烟草 NtGCN2 的原核表达、纯化及多克隆抗体制备[J]. 农业生物技术学报, 2019, 27(1):170-179. doi:10.3969/j.issn.1674-7968.2019.01.018. Hao Y C, Long Y, Guo H, Li N, Zhang S J, Zhao Q, Zhang S T. Prokaryotic expression, purification and polyclonal antibody preparation of tobacco(Nicotiana tabacum) NtGCN2[J]. Journal of Agricultural Biotechnology, 2019, 27(1):170-179.
[26] 蒋琛茜, 瓮巧云, 樊锦涛, 王冠宇, 董丽萍, 邢继红, 董金皋. 拟南芥抗病相关基因 T1N6_22 的原核表达分析[J]. 华北农学报, 2015, 30(1):73-76. doi:10.7668/hbnxb.2015.01.012. Jiang C X, Weng Q Y, Fan J T, Wang G Y, Dong L P, Xing J H, Dong J G. Prokaryotic expression analysis of resistance-related gene T1N6_22 from Arabidopsis thaliana[J]. Acta Agriculturae Boreali-Sinica, 2015, 30(1):73-76.
[27] Khow O, Suntrarachun S. Strategies for production of active eukaryotic proteins in bacterial expression system[J]. Asian Pacific Journal of Tropical Biomedicine, 2012, 2(2):159-162. doi:10.1016/S2221-1691(11)60213-X.
[28] 杨雪, 武志海, 李艳丽, 杨美英. 二穗短柄草 BdGPX4 基因的克隆及酶学性质[J]. 中国农业大学学报, 2018, 23(10):8-17. doi:10.11841/j.issn.1007-4333.2018.10.02. Yang X, Wu Z H, Li Y L, Yang M Y. Cloning and characterization of glutathione peroxidase 4 from Brachypodium distachyon[J]. Journal of China Agricultural University, 2018, 23(10):8-17.
[29] 刘超, 冯勇, 董笑笑, 张忠明, 朱辉. 百脉根泛素结合酶LjE2的表达及活性鉴定[J]. 华中农业大学学报, 2019, 38(1):22-27. doi:10.13300/j.cnki.hnlkxb.2019.01.004. Liu C, Feng Y, Dong X X, Zhang Z M, Zhu H. Expression and activity identification of ubiquitin-binding enzyme LjE2 of Lotus japonicus[J]. Journal of Huazhong Agricultural University, 2019, 38(1):22-27.
[30] 陈伟杰, 付文强, 黄惜, 袁红梅. 橡胶树转录因子HbCBF2和HbCBF3的克隆和原核表达分析[J]. 分子植物育种, 2018, 16(13):4194-4201. doi:10.13271/j.mpb.016.004194. Chen W J, Fu W Q, Huang X, Yuan H M. Cloning and prokaryotic expression of transcription factor HbCBF2 and HbCBF3 in Hevea brasiliensis[J]. Molecular Plant Breeding, 2018, 16(13):4194-4201.

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