Abstract:SnRK2 (sucrose non-fermenting-1-related protein kinase) is a kind of plant-specific Ser/Thr protein kinase, which plays an important role in plant stress resistance. In order to explore the number, structure and expression difference of SnRK2 gene family of strawberry (Fragaria vesca) in response to abiotic stress, based on the conserved sequences of SnRK2 genes in Arabidopsis thaliala and rice (Oryza sativa).Using bioinformatic tools, the homologous comparison, gene structure, systematic evolution, conserved motif, physical and chemical properties of protein, protein secondary structure and sequential action elements of strawberry SnRK2 gene family were analyzed, and qRT-PCR was used to analyze the gene expression under simulated stress. Ten members of SnRK2 gene family were identified from strawberry genome database, divided into 3 subfamilies, the number of coding amino acids is 258~366, and the molecular weight was 29 469.75~41 481.68 D, the theoretical isoelectric points were between 4.68 and 9.10, which were distributed on 4 chromosomes of strawberry. Gene structure and motif analysis showed that most genes had 9 exons, and the distribution of motif in the same subfamily was basically similar. The results of subcellular localization prediction showed that FvSnRK2 gene family was mainly expressed in cytoplasm. The secondary structures of protein were mainly α-helix and irregular curl. Analysis of the upstream 2 kb cis-acting element found that all members of the FvSnRK2 gene family contain MYB and MYC transcription factor response elements. The results of qRT-PCR analysis showed that the relative expression of 5 genes in FvSnRK2 gene family reached the peaks at 12 h after 10 % PEG6000 processing, and the response of FvSnRK2.4 was the most obvious, which was 15 times higher than that at 0 h. After 100 μmol/L ABA (abscisic acid) treatment, the relative expression of 6 genes was the highest at 24 h, and 2 genes had the highest relative expression at 12 h, FvSnRK2.5 and FvSnRK2.9 responsed strongly, which were 16.2 times and 42.4 times higher than that at 0 h, respectively. The relative expression of 7 genes was highest at 2 h after 200 mmol/L NaCl treatment, the response of FvSnRK2.10 was 65.2 times higher than that at 0 h, and FvSnRK2.5 was the highest at 12 h, which was 94.7 times higher than that at 0 h. FvSnRK2.5 and FvSnRK2.10 had strong response after ABA and NaCl treatment, and played important roles in stress resistance signal regulation in strawberry. The present research provides theoretical reference for functional verification and breeding of strawberry SnRK2 gene.
刘涛, 王萍萍, 何红红, 梁国平, 高雪琴, 卢世雄, 陈佰鸿, 毛娟. 草莓SnRK2基因家族的鉴定与表达分析[J]. 农业生物技术学报, 2019, 27(12): 2150-2163.
LIU Tao, WANG Ping-Ping, HE Hong-Hong, LIANG Guo-Ping, GAO Xue-Qin, LU Shi-Xiong, CHEN Bai-Hong, MAO Juan. Identification and Expression Analysis of SnRK2 Gene Family in Strawberry (Fragaria vesca). 农业生物技术学报, 2019, 27(12): 2150-2163.
[1] 敖涛, 胡尊红, 和珊, 等. 2015. 蓖麻SnRK2基因家族的鉴定和特征分析[J]. 中国油料作物学报, 37(2): 160-165. (Ao T, Hu Z H, He S, et al.2015. Identification and characterization of SnRK2 gene family in castor bean (Ricinus communis)[J]. Chinese Journal of Oil Crop Sciences, 37(2): 160-165.) [2] 白戈, 姚恒, 杨大海, 等. 2016. 烟草NtSnRK2基因的鉴定分析[J]. 分子植物育种, 14(10): 2596-2600. (Bai G, Yao H, Yang D H, et al.2016. Characterization of NtSnRK2 gene in tobacco[J]. Molecular Plant Breeding, 14(10): 2596-2600.) [3] 陈娜娜, 刘金义, 蔡斌, 等. 2012. 苹果SnRK2基因家族的鉴定和生物信息学分析[J]. 中国农学通报, 29(13): 120-127. (Chen N N, Liu J Y, Cai B, et al.2012. Identification and bioinformatics analysis of the SnRK2 gene family in apple[J]. Chinese Agricultural Science Bulletin, 29(13): 120-127.) [4] 李利斌, 曹齐卫, 张允楠, 等. 2014. 黄瓜全基因组SnRK2基因的鉴定和序列特征分析[J]. 核农学报, 28(05): 800-807. (Li L B, Cao Q W, Zhang Y N, et al.2014. Identification and sequence analysis of SnRK2 gene genome in cucumber[J]. Journal of Nuclear Agricultural Sciences, 28(05): 800-807.) [5] 李琳, 柳参奎. 2010. SnRK蛋白激酶家族及其成员SnRK2的功能[J]. 分子植物育种, 8(03): 547-555. (Li L, Liu C K.2010. The SnRK protein kinase family and the function of SnRK2 protein kinase[J]. Molecular Plant Breeding, 8(03): 547-555.) [6] 马宗桓, 毛娟, 李文芳, 等. 2016. 葡萄SnRK2家族基因的鉴定与表达分析[J]. 园艺学报, 43(10): 1891-1902. (Ma Z H, Mao J, Li W F, et al.2016. Identification and expression profile of the SnRK2 family genes in grapevine[J]. Acta Horticulturae Sinica, 43(10): 1891-1902.) [7] 王海波. 2016. 小桐子SnRK2基因家族的全基因组鉴定及特征分析[J]. 分子植物育种, 14(9): 2319-2329. (Wang H B.2016. Genome-wide identification and sequence characterization of SnRK2 genes family in Jatropha curcas[J]. Molecular Plant Breeding, 14(9): 2319-2329.) [8] 吴珊, 张欣欣. 2018. 不受ABA诱导的SnRK2蛋白激酶家族研究进展[J]. 基因组学与应用生物学, 37(3): 1364-1369. (Wu S, Zhang X X.2018. Research progress of ABA-independent sucrose non-fermenting protein kinase 2[J]. Genomics and Applied Biology, 37(3): 1364-1369.) [9] 许冰霞, 尹美强, 李娜, 等. 2017. 谷子SnRK2家族基因的生物信息分析[J]. 山西农业大学学报(自然科学版), 37(9): 616.(Xu B X, Yin M Q, Li N, et al. 2017. Identification and bioinformatics analysis of the SnRK2 gene family of Setaria italica[J]. Journal of Shanxi Agricultural University (Natural Science Edition), 37(9): 616.) [10] 颜彦, 丁泽红, 铁韦韦, 等. 2018. 木薯MeSnRK2-1基因克隆及表达分析[J]. 分子植物育种, 16(15): 4839-4844. (Yan Y, Ding Z H, Tie W W, et al.2018. Cloning and expression analysis of MeSnRK2-1 gene in cassava[J]. Molecular Plant Breeding, 16(15): 4839-4844.) [11] Ali G M, Komatsu S.2006. Proteomic analysis of rice leaf sheath during drought stress[J]. Journal of Proteome Research, 5(2): 396-403. [12] Anderberg R J, Walker S M K.1992. Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases[J]. Proceedings of the National Academy of Sciences of the USA, 89(21): 10183-10187. [13] Baradaran A, Sieo C C, Foo H L, et al.2013. Cloning and in silico characterization of two signal peptides from Pediococcus pentosaceus and their function for the secretion of heterologous protein in Lactococcus lactis[J]. Biotechnology Letters, 35: 233-238. [14] Boudsocq M, Barbier-Brygoo H, Laurire C.2004. Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana[J]. Journal of Biological Chemistry, 279(40): 41758-41766. [15] Boudsocq M, Droillard M J, Barbier-Brygoo H, et al.2007. Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid[J]. Plant Molecular Biology, 63(4): 491-503. [16] Cécile Polge P, Thomas M.2007. SNF1/AMPK/SnRK1 kinases, global regulators at the heart of energy control?[J]. Trends in Plant Science, 12(1): 1-28. [17] Cheng C, Wang Z, Ren Z, et al.2017. SCFAtPP2-B11 modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana[J]. PLOS Genetics, 13(8): e1006947. [18] 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 in Arabidopsis[J]. The Plant Cell, 19(2): 485-494. [19] Hadiarto T, Tran L S.2011. Progress studies of drought- responsive genes in rice[J]. Plant Cell Reports, 30(3): 297-310. [20] Halford N G, Boulyz J P, Thomas M.2000. SNF1-related protein kinases (SnRKs)-regulators at the heart of the control of carbon metabolism and partitioning[J]. Advances in Botanical Research, 32(00): 405-434. [21] Halford N G, Hardie D G.1998. SNF1-related protein kinases: Global regulators of carbon metabolism in plants[J]. Plant Molecular Biology, 37: 735-748. [22] Halford N, Hey S.2009. Snf1-related protein kinases (SnRKs) act within an intricate network that links metabolic and stress signalling in plants[J]. Biochemical Journal, 419(2): 247-259. [23] Huai J, Wang M, He J, et al.2008. Cloning and characterization of the SnRK2 gene family from Zea mays[J]. Plant Cell Reports, 27(12): 1861-1868. [24] Kobayashi Y, Yamamoto S, Minami H, et al.2004. Differential activation of the rice sucrose nonfermenting1-related protein kinase 2 family by hyperosmotic stress and abscisic acid[J]. The Plant Cell, 16(5): 1163-1177. [25] Koh S, Lee S C, Kim M K, et al.2007. T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses[J]. Plant Molecular Biology, 65(4): 453-466. [26] 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. [27] Li J, Assmann S M.1996. An abscisic acid-activated and calcium-independent protein kinase from guard cells of fava bean[J]. The Plant Cell Online, 8(12): 2359-2368. [28] Li J, Wang X Q, Watson M B, et al.2000. Regulation of abscisic acid- induced stomatal closure and anion channels by guard cell AAPK kinase[J]. Science, 287: 300-303. [29] Liang S, Wang Y P, Pei C, et al.2011. Transcriptional regulation of SlPYL, SlPP2C and SlSnRK2 gene families encoding ABA signal core components during tomato fruit development and drought stress[J]. Journal of Experimental Botany, 62(15): 5659-5669. [30] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative and the 2-ΔΔCT method[J]. Methods, 25: 402-408. [31] Mao X, Zhang H, Tian S, et al.2010. TaSnRK2.4, an snf1-type serine/threonine protein kinase of wheat (Triticum aestivum L.), confers enhanced multistress tolerance in Arabidopsis[J]. Journal of Experimental Botany, 61(3): 683-696. [32] Monks D E, Aghoram K, Courtney P D, et al.2001. Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2[J].The Plant Cell, 13: 1205-1219. [33] Shin R, Alvarez S, Burch A Y, et al.2007. Phosphoproteomic identification of targets of the Arabidopsis sucrose 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. [34] Shinozaki K, Shinozaki K Y.1997. Gene expression and signal transduction in water-stress response[J]. Plant Physiology, 115(2): 327-334. [35] Singh A, Jha S K, Bagri J, et al.2015. ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis[J]. PLOS ONE, 10(4): e0125168. [36] Song X Q, Yu X, Hori C, et al.2016. Heterologous overexpression of poplar SnRK2 genes enhanced salt stress tolerance in Arabidopsis thaliana[J]. Frontiers in Plant Science, 7: 612-623. [37] Umezawa T, Sugiyama N, Takahashi F, et al.2013. Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in Arabidopsis thaliana[J]. Science Signaling, 6(270): rs8. [38] 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 in Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the USA, 101(49): 17306-17311. [39] Wang Z, Wilson W A, Fujino M A, et al.2001. Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p[J]. Molecular & Cellular Biology, 21(17): 5742-5752. [40] 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 transgenicar Abidopsis[J]. Plant Cell Reports, 30(9): 1683-1699. [41] Yoshida T, Fujita Y, Maruyama K, et al.2015. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress[J]. Plant Cell & Environment, 38(1): 35-49. [42] Yu Q, An L, Li W.2014. The CBL-CIPK network mediates different signaling pathways in plants[J]. Plant Cell Reports, 33: 203-214. [43] Zhang H, Mao X, Wang C, et al.2010. Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis[J]. PLOS ONE, 5: el6041.