小桐子SnRK1蛋白激酶α亚基基因的克隆及原核表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-0948

摘要/Abstract

摘要： 植物蔗糖非发酵-1型相关蛋白激酶1（Sucrose non-fermenting-1 related protein kinase 1,SnRK1）与酵母SNF1及哺乳动物AMPK同属进化上保守的SNF1蛋白激酶家族,参与响应由胞内低能量/低糖状态所引起的信号转导过程。基于小桐子基因组数据,利用生物信息学方法鉴定到小桐子SnRK1蛋白激酶α亚基基因,并克隆到该基因的全长cDNA序列,命名为JcSnRK1α。结果表明,该cDNA序列全长1 700 bp,完整开放阅读框1 545 bp,编码514个氨基酸,分子量为58.8 kD,理论等电点为8.57。基因结构显示,其包含11个外显子与10个内含子。具有包含T-loop区域的激酶结构域、自抑制调控结构域UBA及激酶相关结构域KA1。另外,在该基因启动子序列中鉴定到TATA框、CAAT框及响应高温、低温、干旱、创伤、赤霉素等应答元件。qRT-PCR分析显示,小桐子JcSnRK1α基因具有器官表达特异性,在茎与根中表达量较高,而在叶片中表达量相对较低,同时,在茎与根中都属于低温诱导表达基因,分别在低温胁迫3 h与0.5 h达到最大表达量,较对照提高2.04与3.16倍。构建其原核表达载体pGEX-4T-1-JcSnRK1α,并在Rosetta中诱导表达,得到84.8 kD的蛋白条带,与理论融合蛋白的分子量一致。本研究为开展小桐子JcSnRK1α基因的功能鉴定以及其在信号转导与逆境应答中的机制奠定了基础。

关键词: 小桐子, 蛋白激酶, SnRK1, 基因克隆, 原核表达

Abstract: The SnRK1（Sucrose non-fermenting-1 related protein kinase 1）in plant is ortholog of yeast SNF1（Sucrose non-fermenting-1）and mammalian AMPK（AMP-activated protein kinase）. These evolutionarily conserved kinases are involved in metabolic sensors and cellular signaling caused by energy depletion and low-carbon status. Based on the complete genome data,bioinformatics was used to identify a protein kinase SnRK1 subunit α gene from Jatropha curcas,it was named JcSnRK1α and then cloned and characterized. The results showed that the full-length cDNA of JcSnRK1α was 1700 bp with 11 exons and 10 introns,containing a 1545 bp open reading frame（ORF）. The ORF encoded 514 amino acids with the molecular weight 58.8 kD and the pI value 8.57. Domain analysis by CDD revealed that the JcSnRK1α composed of catalytic kinase domain with T-loop region,together with an ubiquitin-associated domain（UBA）and a kinase-associated domain（KA1）. TATA box,CAAT box,heat,low temperature-,drought-,wound-,and gibberellin-responsive elements were identified in the promoter of JcSnRK1α. qRT-PCR analysis showed that JcSnRK1α expressed specifically in different organs,abundantly in stems and roots,but scarcely in leaves. Meanwhile,JcSnRK1α gene was of remarkably cold-induced expression in stems and roots,which reached the highest after 3 h and 0.5 h chilling stress,respectively,increasing 2.04 and 3.16 times compared to the control. A prokaryotic expression vector pGEX-4T-1-JcSnRK1α was constructed and transformed into Escherichia coli Rosetta,and SDS-PAGE results showed that the molecular weight was 84.8 kD,which was consistent with the expected weight. This study laid a foundation for further studies on the gene function and the mechanism in signaling transduction and stress response in J. curcas.

Key words: Jatropha curcas, protein kinase, SnRK1, gene cloning, prokaryotic expression

王海波, 郭俊云, 田雪莲. 小桐子SnRK1蛋白激酶α亚基基因的克隆及原核表达分析[J]. 生物技术通报, 2019, 35(6): 39-47.

WANG Hai-bo, GUO Jun-yun, TIAN Xue-lian. Molecular Cloning and Prokaryotic Expression Analysis of Protein Kinase SnRK1 Subunit α Gene from Jatropha curcas[J]. Biotechnology Bulletin, 2019, 35(6): 39-47.

参考文献

[1] Makkar HPS, Becker K.Jatropha curcas, a promising crop for the generation of biodiesel and value-added coproducts[J]. European Journal of Lipid Science and Technology, 2009, 111(8):773-787.
[2] Yang CY, Fang Z, Li B, et al.Review and prospects of Jatropha biodiesel industry in China[J]. Renewable & Sustainable Energy Reviews, 2012, 16(4):2178-2190.
[3] 林娟, 周选围, 唐克轩, 等. 麻疯树植物资源研究概况[J]. 热带亚热带植物学报, 2004, 12(3):285-290.
[4] Bohnert HJ, Nelson DE, Jensen RG.Adaptation to environmental stresses[J]. The Plant Cell, 1995, 7(7):1099-1111.
[5] Halford NG, Hey R, et al.Metabolic signalling and carbon partitioning:role of Snf1-related(SnRK1)protein kinase[J]. Journal of Experimental Botany, 2003, 54(382):467-475.
[6] Honigberg SM, Lee RH.Snf1 kinase connects nutritional pathways controlling meiosis in Saccharomyces cerevisiae[J]. Molecular Cell Biology, 1998, 18(8):4548-4555.
[7] Halford NG, Hey S, et al.Highly conserved protein kinases involved in the regulation of carbon and amino acid metabolism[J]. Journal of Experimental Botany, 2004, 55(394):35-42.
[8] Laurie S, McKibbin RS, Halford NG. Antisense SNF1-related(SnRK1)protein kinase gene represses transient activity of an a-amylase(α-AMY2)gene promoter in cultured wheat embryos[J]. Journal of Experimental Botany, 2003, 54(383):739-747.
[9] Harthill JE, Meek SE, Morrice N, et al.Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose[J]. The Plant Journal, 2006, 47(2):211-223.
[10] Dale S, Arro M, Becerra B, et al.Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase(isoform HMGR1)from Arabidopsis thaliana, and its inactivation by phosphorylation at Ser⁵⁷⁷ by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA reductase kinase[J]. European Journal of Biochemistry, 1995, 233(2):506-513.
[11] Sugden C, Donaghy PG, Halford NG, et al.Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro[J]. Plant Physiology, 1999, 120(1):257-274.
[12] Hanson J, Hanssen M, Wiese A, et al.The sucrose regulated transcription factor bZIP11 affects amino acid metabolism by regulating the expression of asparagine synthetase 1 and proline dehydrogenase 2[J]. Plant Journal, 2008, 53(6):935-949.
[13] Holappa LD, Walker-Simmons MK, Ho THD, et al.A Triticum tauschii protein kinase related to wheat PKABA1 is associated with ABA signaling and is distributed between the nucleus and cytosol[J]. Journal of Cereal Science, 2005, 41(3):333-346.
[14] Geigenberger P.Regulation of sucrose to starch conversion in growing potato tubers[J]. Journal of Experimental Botany, 2003, 54(328):457-465.
[15] Lovas A, Bimbo A, Szabo L, et al.Antisense repression of StubGAL83 affects root and tuber development in potato[J]. The Plant Journal, 2003, 33(1):139-147.
[16] Hao L, Wang H, Sunter G, et al.Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase[J]. The Plant Cell, 2003, 15(4):1034-1048.
[17] Alderson A, Sabelli PA, Dickinson JR, et al.Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA[J]. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(19):8602-8605.
[18] Tome F, Nagele T, Adamo M, et al.The low energy signaling network[J]. Frontiers in Plant Science, 2014, 5(5):353.
[19] Crozet P, Margalha L, Confraria A, et al.Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases[J]. Frontiers in Plant Science, 2014, 5(2):190.
[20] Ghillebert R, Swinnen E, Wen J, et al.The AMPK/SNF1/SnRK1 fuel gauge and energy regulator:structure, function and regulation[J]. The FEBS Journal, 2011, 278(21):3978-3990.
[21] Jiang R, Carlson M.The Snf1 protein kinsae and its activating subunit Snf4 interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex[J]. Molecular Cell Biolology, 1997, 17(4):2099-2106.
[22] Baena-Gonzalez E, Rolland F, Thevelein JM, et al.A central integrator of transcription networks in plant stress and energy signaling[J]. Nature, 2007, 448(7156):938-942.
[23] Tsai AYL, Gazzarrini S.Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling:the emerging picture[J]. Frontiers in Plant Science, 2014, 5(5):119.
[24] Lunn JE, Delorge I, Figueroa C, et al.Trehalose metabolism in plants[J]. Plant Journal for Cell & Molecular Biology, 2014, 79(4):544-567.
[25] Lu CA, Lin C, Wei K, et al.The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice[J]. Plant Cell, 2007, 19(8):2484-2499.
[26] Nunes C, Primavesi LF, Patel MK, et al.Inhibition of SnRK1 by metabolites:tissue-dependent effects and cooperative inhibition by glucose 1-phosphate in combination with trehalose 6-phosphate[J]. Plant Physiology and Biochemistry, 2013, 63(4):89-98.
[27] Shen W, Reyes MI, et al.Arabidopsis protein kinases GRIK1 and
GRIK2 specifically activate SnRK1 by phosphorylating its activa-tion Loop[J]. Plant Physiology, 2009, 150(2):996-1005.
[28] Tsai AYL, Gazzarrini S.AKIN10 and FUSCA3 interact to control lateral organ development and phase transitions in Arabidopsis[J]. Plant Journal for Cell & Molecular Biology, 2012, 69(5):809-821.
[29] Confraria A, Martinho C, Elias A, et al.miRNAs mediate SnRK1-dependent energy signalling in Arabidopsis[J]. Frontiers in Plant Science, 2013, 4(1):197.
[30] Machintosh RW, Davies SP, Clarike PR, et al.Evidence for a protein kinase cascade in higher plants 3-hydroxy-3-methylglutaryl-CoA reductase kinase[J]. European Journal of Biochemistry, 1992, 209(3):923-931.
[31] Radchuk R, Radchuk V, Weschke W, et al.Repressing the expression of the sucrose non fermenting-1-related protein kinase gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype[J]. Plant Physiology, 2006, 140(1):263-278.
[32] Zhang Y, Shewry PR, et al.Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley[J]. Plant Journal, 2001, 28(4):431-441.
[33] Kleinow T, Himbert S, Krenz B, et al.NAC domain transcription factor ATAF1 interacts with SNF1-related kinases and silencing of its subfamily causes severe developmental defects in Arabidopsis[J]. Plant Science, 2009, 177(4):360-370.