橡胶树HbSnRK2.6家族基因的克隆、原核表达及纯化
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摘要
低温是重要的环境胁迫因子,严重影响中国橡胶树的分布及胶乳的产量。在拟南芥中,低温能够激活蛋白激酶SnRK2.6,并提高植物耐寒性能,但橡胶树中的SnRK2.6至今未被克隆。本研究以橡胶树7-33-97为材料,从cDNA中扩增出两个SnRK2.6基因(HbSnRK2.6A和HbSnRK2.6B)。氨基酸序列比对分析表明,Hb SnRK2.6A和HbSnRK2.6B与其他物种中的SnRK2.6存在较高的同源性。将HbSnRK2.6A及HbSnRK2.6B构建到原核表达载体上,在E. coli (DE3)中诱导并纯化HbSnRK2.6重组蛋白,结果表明HbSnRK2.6A和HbSnRK2.6B的最佳诱导条件是:16℃诱导16 h,IPTG浓度为0.6 mmol/L。最终我们成功在大肠杆菌中表达并得到了大量橡胶树中的蛋白激酶SnRK2.6。
Low temperature stress is one of the most devastating environmental factors adversely affecting rubber geographic distribution and latex productivity. It has been documented that SnRK2.6 kinase increased freezing tolerance in Arabidopsis, but SnRK2.6 in Hevea brasiliensis has not been cloned so far. In this study, two SnRK2.6 genes, SnRK2.6 A and SnRK2.6 B were cloned from Reyan 7-33-97 rubber trees. Multiple alignments of HbSnRK2.6 A/Hb Sn RK2.6 B and SnRK2.6 proteins from other plants indicated that all SnRK2.6 proteins tested were highly conserved. HbSnRK2.6 A and HbSnRK2.6 B were constructed into prokaryotic expression vectors to induce and purify Hb Sn RK2.6 recombinant proteins in E. coli(DE3). Our results showed that the optimal induction conditions for Hb Sn RK2.6 A/HbSnRK2.6 B recombinant proteins were 0.6 mmol/L IPTG at 16℃ for 16 h. In the end,we got a large number of protein kinase SnRK2.6 of Hevea brasiliensis from E. coli.
Low temperature stress is one of the most devastating environmental factors adversely affecting rubber geographic distribution and latex productivity. It has been documented that SnRK2.6 kinase increased freezing tolerance in Arabidopsis, but SnRK2.6 in Hevea brasiliensis has not been cloned so far. In this study, two SnRK2.6 genes, SnRK2.6 A and SnRK2.6 B were cloned from Reyan 7-33-97 rubber trees. Multiple alignments of HbSnRK2.6 A/Hb Sn RK2.6 B and SnRK2.6 proteins from other plants indicated that all SnRK2.6 proteins tested were highly conserved. HbSnRK2.6 A and HbSnRK2.6 B were constructed into prokaryotic expression vectors to induce and purify Hb Sn RK2.6 recombinant proteins in E. coli(DE3). Our results showed that the optimal induction conditions for Hb Sn RK2.6 A/HbSnRK2.6 B recombinant proteins were 0.6 mmol/L IPTG at 16℃ for 16 h. In the end,we got a large number of protein kinase SnRK2.6 of Hevea brasiliensis from E. coli.
引文
Agarwal M.,Hao Y.,Kapoor A.,Dong C.H.,Fujii H.,Zheng X.,and Zhu J.K.,2006,A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance,J.Biol.Chem.,281(49):37636-37645
Boudsocq M.,Barbier-Brygoo H.,and Lauriere C.,2004,Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana,J.Biol.Chem.,279(40):41758-41766
Catala R.,Santos E.,Alonso J.M.,Ecker J.R.,Martinez-Zapater J.M.,and Salinas J.,2003,Mutations in the Ca2+/H+transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis,Plant Cell,15(12):2940-2951
Cheng H.,Cai H.,Fu H.,An Z.,Fang J.,Hu Y.,Guo D.,and Huang H.,2015,Functional characterization of Hevea brasiliensis CRT/DRE binding factor 1 gene revealed regulation potential in the CBF Pathway of tropical perennial tree,PLoS One,10(9):e0137634
Chinnusamy V.,Schumaker K.,and Zhu J.K.,2004,Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants,J.Exp.Bot.,55(395):225-236
Ding Y.,Li H.,Zhang X.,Xie Q.,Gong Z.,and Yang S.,2015,SnRK2 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis,Dev.Cell,32(3):278-289
Dong C.H.,Agarwal M.,Zhang Y.,Xie Q.,and 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,Proc.Natl.Acad.Sci.USA,103(21):8281-8286
Farras R.,Ferrando A.,Jasik J.,Kleinow T.,Okresz L.,Tiburcio A.,Salchert K.,del Pozo C.,Schell J.,and Koncz C.,2001,SKP1-SnRK protein kinase interactions mediate proteasomal binding of a pla nt SCF ubiquitin ligase,EMBO J.,20(11):2742-2756
Halford N.G.,Hey S.,Jhurreea D.,Laurie S.,McKibbin R.S.,Paul M.,and Zhang Y.,2003,Metabolic signalling and carbon partitioning:role of Snf1-related(SnRK1)protein kinase,J.Exp.Bot.,54(382):467-475
Huai J.,Wang M.,He J.,Zheng J.,Dong Z.,Lv H.,Zhao J.,and Wang G.,2008,Cloning and characterization of the SnRK2gene family from Zea mays,Plant Cell Rep.,27(12):1861-1868
Joshi-Saha A.,Valon C.,and Leung J.,2011,Abscisic acid signal off the STARting block,Mol.Plant,4(4):562-580
Li X.,Wang J.,Ding B.,Wu T.W.,Yu P.,Chen X.Q.,and Xie X.D.,2017,Advances in the regulation of OST1 on stomatal movement and stress responses of plant,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(2):834-840(李欣,王俊斌,丁博,吴天文,余鹏,陈小强,谢晓东,2017,OST1调控植物气孔运动和逆境响应的研究进展,基因组学与应用生物学,36(2):834-840)
Mao X.,Zhang H.,Tian S.,Chang X.,and Jing R.,2010,TaS-nRK2.4,an SNF1-type serine/threonine protein kinase of wheat(Triticum aestivum L.),confers enhanced multistress tolerance in Arabidopsis,J.Exp.Bot.,61(3):683-696
Mickelbart M.V.,Hasegawa P.M.,and Bailey-Serres J.,2015,Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability,Nat.Rev.Genet.,16(4):237-251
Moffatt B.,Ewart V.,and Eastman A.,2010,Cold comfort:plant antifreeze proteins,Physiol.Plantarum,126(1):5-16
Nakashima K.,Fujita Y.,Kanamori N.,Katagiri T.,Umezawa T.,Kidokoro S.,Maruyama K.,Yoshida T.,Ishiyama K.,Kobayashi M.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2009,Three Arabidopsis SnRK2 protein kinases,SRK2D/SnRK2.2,SRK2E/SnRK2.6/SNRK2A and SRK2I/SnRK2.3,involved in ABA signaling are essential for the control of seed development and dormancy,Plant Cell Physiol.,50(7):1345-1363
Ryu J.Y.,Hong S.Y.,Jo S.H.,Woo J.C.,Lee S.,and Park C.M.,2014,Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon,BMC Plant Biol.,14:1471-2229
Shi Y.T.,Tian S.W.,Hou L.Y.,Huang X.Z.,Zhang X.Y.,Guo H.W.,and Yang S.H.,2012,Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBFand Type-A ARR genes in Arabidopsis,Plant Cell,24(6):2578-2595
Sirichandra C.,Davanture M.,Turk B.E.,Zivy M.,Valot B.,Leung J.,and Merlot S.,2010,The Arabidopsis ABA-activated kinase SNRK2A phosphorylates the bZIP transcription factor ABF3 and creates a 14-3-3 binding site involved in its turnover,PLoS One,5(11):e13935
Thomashow M.F.,1998,Role of cold-responsive genes in plant freezing tolerance,Plant Physiol.,118(1):1-7
Umezawa T.,Sugiyama N.,Takahashi F.,Anderson J.C.,Ishihama Y.,Peck S.C.,and Shinozaki K.,2013,Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in Arabidopsis thaliana,Sci.Signal.,6(270):rs8
Vilela B.,Moreno-Cortes A.,Rabissi A.,Leung J.,Pages M.,and Lumbreras V.,2013,The maize SNRK2A kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor,PLoS One,8(2):e58105
Wang M.,Yuan F.,Hao H.,Zhang Y.,Zhao H.,Guo A.,Hu J.,Zhou X.,and Xie C.G.,2013a,BolSNRK2A,an ortholog of open stomata 1 with alternative splicing products in Brassica oleracea,positively modulates drought responses in plants,Biochem.Biophys Res.Commun.,442(3-4):214-220
Wang P.,Xue L.,Batelli G.,Lee S.,Hou Y.J.,Van Oosten M.J.,Zhang H.,Tao W.A.,and Zhu J.K.,2013b,Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action,Proc.Natl.Acad.Sci.USA,110(27):11205-11210
Wang Q.,Guo Q.,Guo Y.,Yang J.,Wang M.,Duan X.,Niu J.,Liu S.,Zhang J.,and Lu Y.,2018,Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance by interacting with OST1,J.Exp.Bot.,69(18):4403-4417
Wu J.,Yamagata H.,Hayashi-Tsugane M.,Hijishita S.,Fujisawa M.,Shibata M.,Ito Y.,Nakamura M.,Sakaguchi M.,Yoshihara R.,Kobayashi H.,Ito K.,Karasawa W.,Yamamoto M.,Saji S.,Katagiri S.,Kanamori H.,Namiki N.,Katayose Y.,Matsumoto T.,and Sasaki T.,2004,Composition and structure of the centromeric region of rice chromosome 8,Plant Cell,16(4):967-976
Wu S.,and Zhang X.,2018,Research progress of ABA-independent sucrose non-fermenting protein kinase 2,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(3):1364-1369(吴珊,张欣欣,2018,不受ABA诱导的SnRK2蛋白激酶家族研究进展,基因组学与应用生物学,37(3):1364-1369)
Xue S.,Hu H.,Ries A.,Merilo E.,Kollist H.,and Schroeder J.I.,2011,Central functions of bicarbonate in S-type anion channel activation and SNRK2A protein kinase in CO2signal transduction in guard cell,EMBO J.,30(8):1645-1658
Yuan H.M.,Sheng Y.,Chen W.J.,Lu Y.Q.,Tang X.,Ou-Yang M.,and Huang X.,2017,Overexpression of Hevea brasiliensis HbICE1 enhances cold tolerance in Arabidopsis,Front Plant Sci.,8:1462
Zheng Z.,Xu X.,Crosley R.A.,Greenwalt S.A.,Sun Y.,Blakeslee B.,Wang L.,Ni W.,Sopko M.S.,Yao C.,Yau K.,Burton S.,Zhuang M.,McCaskill D.G.,Gachotte D.,Thompson M.,and Greene T.W.,2010,The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis,Plant Physiol.,153(1):99-113
Boudsocq M.,Barbier-Brygoo H.,and Lauriere C.,2004,Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana,J.Biol.Chem.,279(40):41758-41766
Catala R.,Santos E.,Alonso J.M.,Ecker J.R.,Martinez-Zapater J.M.,and Salinas J.,2003,Mutations in the Ca2+/H+transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis,Plant Cell,15(12):2940-2951
Cheng H.,Cai H.,Fu H.,An Z.,Fang J.,Hu Y.,Guo D.,and Huang H.,2015,Functional characterization of Hevea brasiliensis CRT/DRE binding factor 1 gene revealed regulation potential in the CBF Pathway of tropical perennial tree,PLoS One,10(9):e0137634
Chinnusamy V.,Schumaker K.,and Zhu J.K.,2004,Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants,J.Exp.Bot.,55(395):225-236
Ding Y.,Li H.,Zhang X.,Xie Q.,Gong Z.,and Yang S.,2015,SnRK2 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis,Dev.Cell,32(3):278-289
Dong C.H.,Agarwal M.,Zhang Y.,Xie Q.,and 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,Proc.Natl.Acad.Sci.USA,103(21):8281-8286
Farras R.,Ferrando A.,Jasik J.,Kleinow T.,Okresz L.,Tiburcio A.,Salchert K.,del Pozo C.,Schell J.,and Koncz C.,2001,SKP1-SnRK protein kinase interactions mediate proteasomal binding of a pla nt SCF ubiquitin ligase,EMBO J.,20(11):2742-2756
Halford N.G.,Hey S.,Jhurreea D.,Laurie S.,McKibbin R.S.,Paul M.,and Zhang Y.,2003,Metabolic signalling and carbon partitioning:role of Snf1-related(SnRK1)protein kinase,J.Exp.Bot.,54(382):467-475
Huai J.,Wang M.,He J.,Zheng J.,Dong Z.,Lv H.,Zhao J.,and Wang G.,2008,Cloning and characterization of the SnRK2gene family from Zea mays,Plant Cell Rep.,27(12):1861-1868
Joshi-Saha A.,Valon C.,and Leung J.,2011,Abscisic acid signal off the STARting block,Mol.Plant,4(4):562-580
Li X.,Wang J.,Ding B.,Wu T.W.,Yu P.,Chen X.Q.,and Xie X.D.,2017,Advances in the regulation of OST1 on stomatal movement and stress responses of plant,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(2):834-840(李欣,王俊斌,丁博,吴天文,余鹏,陈小强,谢晓东,2017,OST1调控植物气孔运动和逆境响应的研究进展,基因组学与应用生物学,36(2):834-840)
Mao X.,Zhang H.,Tian S.,Chang X.,and Jing R.,2010,TaS-nRK2.4,an SNF1-type serine/threonine protein kinase of wheat(Triticum aestivum L.),confers enhanced multistress tolerance in Arabidopsis,J.Exp.Bot.,61(3):683-696
Mickelbart M.V.,Hasegawa P.M.,and Bailey-Serres J.,2015,Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability,Nat.Rev.Genet.,16(4):237-251
Moffatt B.,Ewart V.,and Eastman A.,2010,Cold comfort:plant antifreeze proteins,Physiol.Plantarum,126(1):5-16
Nakashima K.,Fujita Y.,Kanamori N.,Katagiri T.,Umezawa T.,Kidokoro S.,Maruyama K.,Yoshida T.,Ishiyama K.,Kobayashi M.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2009,Three Arabidopsis SnRK2 protein kinases,SRK2D/SnRK2.2,SRK2E/SnRK2.6/SNRK2A and SRK2I/SnRK2.3,involved in ABA signaling are essential for the control of seed development and dormancy,Plant Cell Physiol.,50(7):1345-1363
Ryu J.Y.,Hong S.Y.,Jo S.H.,Woo J.C.,Lee S.,and Park C.M.,2014,Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon,BMC Plant Biol.,14:1471-2229
Shi Y.T.,Tian S.W.,Hou L.Y.,Huang X.Z.,Zhang X.Y.,Guo H.W.,and Yang S.H.,2012,Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBFand Type-A ARR genes in Arabidopsis,Plant Cell,24(6):2578-2595
Sirichandra C.,Davanture M.,Turk B.E.,Zivy M.,Valot B.,Leung J.,and Merlot S.,2010,The Arabidopsis ABA-activated kinase SNRK2A phosphorylates the bZIP transcription factor ABF3 and creates a 14-3-3 binding site involved in its turnover,PLoS One,5(11):e13935
Thomashow M.F.,1998,Role of cold-responsive genes in plant freezing tolerance,Plant Physiol.,118(1):1-7
Umezawa T.,Sugiyama N.,Takahashi F.,Anderson J.C.,Ishihama Y.,Peck S.C.,and Shinozaki K.,2013,Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in Arabidopsis thaliana,Sci.Signal.,6(270):rs8
Vilela B.,Moreno-Cortes A.,Rabissi A.,Leung J.,Pages M.,and Lumbreras V.,2013,The maize SNRK2A kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor,PLoS One,8(2):e58105
Wang M.,Yuan F.,Hao H.,Zhang Y.,Zhao H.,Guo A.,Hu J.,Zhou X.,and Xie C.G.,2013a,BolSNRK2A,an ortholog of open stomata 1 with alternative splicing products in Brassica oleracea,positively modulates drought responses in plants,Biochem.Biophys Res.Commun.,442(3-4):214-220
Wang P.,Xue L.,Batelli G.,Lee S.,Hou Y.J.,Van Oosten M.J.,Zhang H.,Tao W.A.,and Zhu J.K.,2013b,Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action,Proc.Natl.Acad.Sci.USA,110(27):11205-11210
Wang Q.,Guo Q.,Guo Y.,Yang J.,Wang M.,Duan X.,Niu J.,Liu S.,Zhang J.,and Lu Y.,2018,Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance by interacting with OST1,J.Exp.Bot.,69(18):4403-4417
Wu J.,Yamagata H.,Hayashi-Tsugane M.,Hijishita S.,Fujisawa M.,Shibata M.,Ito Y.,Nakamura M.,Sakaguchi M.,Yoshihara R.,Kobayashi H.,Ito K.,Karasawa W.,Yamamoto M.,Saji S.,Katagiri S.,Kanamori H.,Namiki N.,Katayose Y.,Matsumoto T.,and Sasaki T.,2004,Composition and structure of the centromeric region of rice chromosome 8,Plant Cell,16(4):967-976
Wu S.,and Zhang X.,2018,Research progress of ABA-independent sucrose non-fermenting protein kinase 2,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(3):1364-1369(吴珊,张欣欣,2018,不受ABA诱导的SnRK2蛋白激酶家族研究进展,基因组学与应用生物学,37(3):1364-1369)
Xue S.,Hu H.,Ries A.,Merilo E.,Kollist H.,and Schroeder J.I.,2011,Central functions of bicarbonate in S-type anion channel activation and SNRK2A protein kinase in CO2signal transduction in guard cell,EMBO J.,30(8):1645-1658
Yuan H.M.,Sheng Y.,Chen W.J.,Lu Y.Q.,Tang X.,Ou-Yang M.,and Huang X.,2017,Overexpression of Hevea brasiliensis HbICE1 enhances cold tolerance in Arabidopsis,Front Plant Sci.,8:1462
Zheng Z.,Xu X.,Crosley R.A.,Greenwalt S.A.,Sun Y.,Blakeslee B.,Wang L.,Ni W.,Sopko M.S.,Yao C.,Yau K.,Burton S.,Zhuang M.,McCaskill D.G.,Gachotte D.,Thompson M.,and Greene T.W.,2010,The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis,Plant Physiol.,153(1):99-113
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