Cloning and Expressional Analysis Under Abiotic Stress of the Protein Kinase Gene AsSnRK2.10 in Oat (Avena sativa)
XIANG Dian-Jun1, MAN Li-Li2*, WANG Qing-Xiang1, WANG Xiao-Dong1, ZHANG Wei-Wei1, LIU Peng1, LI Zhi-Gang1
1. College of Agriculture,Inner Mongolia University for Nationalities,Tongliao 028042,China; 2. College of Life Sciences and Food Engineering,Inner Mongolia University for Nationalities,Tongliao 028042,China
Abstract：Sucrose non-fermented related protein kinase 2 (SnRK2) family members are plant-specific serine/threonine kinases involved in response to abiotic stresses. In this study,to explore the biological roles of SnRK2 gene in growth,development,and abiotic stress tolerance in oat (Avena sativa),one of the SnRK2 family members,designated AsSnRK2.10 (GenBank No. MN729578),was isolated from oat variety 'Monida' by reverse transcription-PCR (RT-PCR). The molecular characteristics of AsSnRK2.10 encoded protein were predicted by bioinformatics analysis. Then subcellular localization of target protein was performed using a transient expression system in tobacco (Nicotiana benthamiana). And lastly,the expression profiles of AsSnRK2.10 in different tissues and under abiotic stress conditions were detected by qRT-PCR,respectively. The results showed that AsSnRK2.10 gene contained a 1 086 bp of open reading frame (ORF),which encoded a polypeptide of 361 amino acid polypeptide with a predicted molecular weight of 40.66 kD and a theoretical isoelectric point of 4.75. The AsSnRK2.10 protein contained the SnRK2-specific functional domains,including a protein kinase adenosine triphosphate (ATP)-binding signaling region,a N-myristoylation site,a serine/threonine protein kinase active site,an activation loop,a transmembrane helix site,a domain 1 required for the osmotic stress response,and a domain 2 required for the abscisic acid (ABA) response. The smallest secondary structure element of the AsSnRK2.10 protein was beta turn,and the largest was random coil. Phylogenetic analysis showed that the AsSnRK2.10 protein had the closest evolutionary relationship with SnRK2 members from seven species,and was clustered into one branch. The subcellular localization analysis revealed that the AsSnRK2.7 protein was localized primarily in the nucleus. Tissue-specific expression showed that AsSnRK2.10 was expressed in the roots,stems,leaves,and panicles of oat during different development stages. However,the highest transcript levels of the AsSnRK2.10 gene in the roots,stems,leaves,and panicles occurred at the heading stage,filling stage,tillering stage,and heading stage,respectively. Furthermore,AsSnRK2.10 gene was involved in ABA signal transduction and positively responded to dehydration,high salinity,and low-temperature stresses. The results provide a theoretical basis for further study on the biological function of AsSnRK2.10 gene and its application in oat stress resistance breeding.
向殿军, 满丽莉, 王庆祥, 王晓东, 张巍巍, 刘鹏, 李志刚. 燕麦蛋白激酶基因AsSnRK2.10的克隆及非生物胁迫响应分析[J]. 农业生物技术学报, 2020, 28(11): 1923-1935.
XIANG Dian-Jun, MAN Li-Li, WANG Qing-Xiang, WANG Xiao-Dong, ZHANG Wei-Wei, LIU Peng, LI Zhi-Gang. Cloning and Expressional Analysis Under Abiotic Stress of the Protein Kinase Gene AsSnRK2.10 in Oat (Avena sativa). 农业生物技术学报, 2020, 28(11): 1923-1935.
[1] 刘涛,王萍萍,何红红,等.2019.草莓SnRK2基因家族的鉴定与表达分析[J].农业生物技术学报,27(12):2150-2163. (Liu T,Wang P P,He H H,et al.2019,Identification and Expression Analysis ofSnRK2Gene Family in Strawberry (Fragaria vesca)[J].Journal of Agricultural Biotechnology,27(12):2150-2163.) [2] Bakhshandeh E,Gholamhosseini M,Yaghoubian Y,et al.2020.Plant growth promoting microorganisms can improve germination,seedling growth and potassium uptake of soybean under drought and salt stress[J].Plant Growth Regulation,90(1):123-136. [3] Boudsocq M,Barbier-Brygoo H,Laurière C.2004.Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses inArabidopsis thaliana[J].Journal of Biological Chemistry,279(40):41758-41766. [4] Burza A M,Pękala I,Sikora J,et al.2006.Nicotiana tabacumosmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop[J].Journal of Biological Chemistry,281(45):34299-34311. [5] Diédhiou C J,Popova O V,Dietz K J,et al.2008.The SNF1-type serine-threonine protein kinaseSAPK4regulates stress-responsive gene expression in rice[J].BMC Plant Biology,8(1):49. [6] Fujii H,Verslues P E,Zhu J K.2007.Identification of two protein kinases required for abscisic acid regulation of seed germination,root growth,and gene expression inArabidopsis[J].The Plant Cell,19(2):485-494. [7] Fujita Y,Nakashima K,Yoshida T,et al.2009.Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress inArabidopsis[J].Plant and Cell Physiology,50(12):2123-2132. [8] Fujita Y,Yoshida T,Yamaguchi‐Shinozaki K.2013.Pivotal role of the AREB/ABF‐SnRK2 pathway in ABRE‐mediated transcription in response to osmotic stress in plants[J].Physiologia Plantarum,147(1):15-27. [9] Furihata T,Maruyama K,Fujita Y,et al.2006.Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1[J].Proceedings of the National Academy of Sciences of the USA,103(6):1988-1993. [10] Huai J,Wang M,He J,et al.2008.Cloning and characterization of the SnRK2 gene family fromZea mays[J].Plant Cell Reports,27(12):1861-1868. [11] Huang C,Verrillo F,Renzone G,et al.2011.Response to biotic and oxidative stress inArabidopsis thaliana:Analysis of variably phosphorylated proteins[J].Journal of Proteomics,74(10):1934-1949. [12] Ishitani M,Liu J,Halfter U,et al.2000.SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding[J].The Plant Cell,12(9):1667-1677. [13] Kobayashi Y,Yamamoto S,Minami H,et al.2004.Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid[J].The Plant Cell,16(5):1163-1177. [14] Kulik A,Wawer I,Krzywińska E,et al.2011.SnRK2 protein kinases-key regulators of plant response to abiotic stresses[J].Omics:A Journal of Integrative Biology,15(12):859-872. [15] Laurie S,Halford N G.2001.The role of protein kinases in the regulation of plant growth and development[J].Plant Growth Regulation,34(3):253-265. [16] Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,25(4):402-408. [17] Mahajan S,Tuteja N.2005.Cold,salinity and drought stresses:an overview[J].Archives of Biochemistry and Biophysics,444(2):139-158. [18] 燕麦蛋白激酶基因AsSnRK2.10的克隆及非生物胁迫响应分析 Cloning and Expressional Analysis Under Abiotic Stress of the Protein Kinase GeneAsSnRK2.10in Oat(Avena sativa) [19] Mao X,Zhang H,Tian S,et al.2010.TaSnRK2.4,an SNF1-type serine/threonine protein kinase of wheat (Triticum aestivumL.),confers enhanced multistress tolerance inArabidopsis[J].Journal of Experimental Botany,61(3):683-696. [20] Mizoguchi M,Umezawa T,Nakashima K,et al.2010.Two closely related subclass II SnRK2 protein kinases cooperatively regulate drought-inducible gene expression[J].Plant and Cell Physiology,51(5):842-847. [21] Nakashima K,Fujita Y,Kanamori N,et al.2009.ThreeArabidopsisSnRK2 protein kinases,SRK2D/SnRK2.2,SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3,involved in ABA signaling are essential for the control of seed development and dormancy[J].Plant and Cell Physiology,50(7):1345-1363. [22] Park S Y,Fung P,Nishimura N,et al.2009.Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins[J].Science,324(5930):1068-1071. [23] Sheard L B,Zheng N.2009.Plant biology:Signal advance for abscisic acid[J].Nature,462(7273):575. [24] Sheludko Y V,Sindarovska Y R,Gerasymenko I M,et al.2007.Comparison of severalNicotianaspecies as hosts for high‐scaleAgrobacterium‐mediated transient expression[J].Biotechnology and Bioengineering,96(3):608-614. [25] Shin R,Alvarez S,Burch A Y,et al.2007.Phosphoproteomic identification of targets of theArabidopsissucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes[J].Proceedings of the National Academy of Sciences of the USA,104(15):6460-6465. [26] Tian S,Mao X,Zhang H,et al.2013.Cloning and characterization ofTaSnRK2.3,a novelSnRK2gene in common wheat[J].Journal of Experimental Botany,64(7):2063-2080. [27] Torres L F,Reichel T,Déchamp E,et al.2019.Torres L F,Reichel T,Déchamp E,et al.Expression ofDREB-like genes inCoffea canephoraandC.arabicasubjected to various types of abiotic stress[J].Tropical Plant Biology,12(2):98-116. [28] Umezawa T,Nakashima K,Miyakawa T,et al.2010.Molecular basis of the core regulatory network in ABA responses:Sensing,signaling and transport[J].Plant and Cell Physiology,51(11):1821-1839. [29] Umezawa T,Yoshida R,Maruyama K,et al.2004.SRK2C,a SNF1-related protein kinase 2,improves drought tolerance by controlling stress-responsive gene expression inArabidopsis thaliana[J].Proceedings of the National Academy of Sciences of the USA,101(49):17306-17311. [30] Vlad F,Droillard M J,Valot B,et al.2010.Phospho‐site mapping,genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s[J].The Plant Journal,63(5):778-790. [31] Wang L,Hu W,Sun J,et al.2015.Genome-wide analysis ofSnRKgene family inBrachypodium distachyonand functional characterization ofBdSnRK2.9[J].Plant Science,237:33-45. [32] Wang P,Xue L,Batelli G,et al.2013.Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action[J].Proceedings of the National Academy of Sciences of the USA,110(27):11205-11210. [33] Wang R,Wang C,Yao Q,et al.2019.The polish wheat (Triticum polonicumL.)TpSnRK2.10andTpSnRK2.11meditate the accumulation and the distribution of cd and Fe in transgenic Arabidopsis plants[J].BMC Genomics,20(1):210. [34] Wei H,Movahedi A,Xu C,et al.2019.Heterologous overexpression of theArabidopsis SnRK2.8gene enhances drought and salt tolerance inPopulus × euramericana cv.'Nanlin895'[J].Plant Biotechnology Reports,13(3):245-261. [35] Xiang D J,Man L L,Cao S,et al.2020.Heterologous expression of anAgropyron cristatumSnRK2 protein kinase gene (AcSnRK2.11) increases freezing tolerance in transgenic yeast and tobacco[J].3 Biotech,10(5):209. [36] Xiong L,Schumaker K S,Zhu J K.2002.Cell signaling during cold,drought,and salt stress[J].The Plant Cell,14(suppl 1):S165-S183. [37] Ying S,Zhang D F,Li H Y,et al.2011.Cloning and characterization of a maize SnRK2 protein kinase gene confers enhanced salt tolerance in transgenicArabidopsis[J].Plant Cell Reports,30(9):1683-1699. [38] Yoshida T,Mogami J,Yamaguchi-Shinozaki K.2014.ABA-dependent and ABA-independent signaling in response to osmotic stress in plants[J].Current Opinion in Plant Biology,21:133-139. [39] Zhang H,Mao X,Jing R,et al.2011.Characterization of a common wheat (Triticum aestivumL.)TaSnRK2.7gene involved in abiotic stress responses[J].Journal of Experimental Botany,62(3):975-988. [40] Zhang H,Mao X,Wang C,et al.2010.Overexpression of a common wheat geneTaSnRK2.8enhances tolerance to drought,salt and low temperature inArabidopsis[J].PLOS ONE,5(12):e16041. [41] Zhu J K.2002.Salt and drought stress signal transduction in plants[J].Annual Review of Plant Biology,53(1):247-273.
