草地早熟禾蛋白激酶<i>SnRK2.4</i> 基因克隆及非生物胁迫响应分析

doi:10.7668/hbnxb.20193082

摘要/Abstract

摘要：

蛋白激酶是植物逆境防御机制中重要的核心成分,植物蛋白激酶SnRK2是一种丝氨酸/苏氨酸蛋白激酶,可通过磷酸化途径在植物逆境胁迫响应中发挥重要作用。分析草地早熟禾蛋白激酶基因SnRK2.4在非生物胁迫下的表达规律,旨在揭示其在逆境调控中的作用,以优质的冷季型草坪草草地早熟禾为试材,利用RT-PCR方法克隆得到SnRK2.4基因,其包含一个1 092 bp开放阅读框区,编码363个氨基酸序列,利用生物信息学分析其分子特征,并采用实时荧光定量PCR方法观测不同组织部位、非生物胁迫下该基因表达模式。结果表明,草地早熟禾SnRK2.4属于SRK/SAPK 家族,包含典型的STKc_SnRK2结构域,酪氨酸激酶磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、丝氨酸/苏氨酸蛋白激酶活性位点等功能域,其与二穗短柄草同源性最高。实时荧光定量PCR结果表明,草地早熟禾SnRK2.4基因表达水平存在组织特异性,穗部表达量最高,根、茎、叶中无显著差异,草地早熟禾SnRK2.4基因积极响应干旱、盐、低氮、低磷、ABA、BR等非生物胁迫。

关键词: 草地早熟禾, 蛋白激酶, SnRK2.4, 基因克隆, 非生物胁迫

Abstract:

Protein kinases are important factors in plant defense system,protein kinase SnRK2 is a serine/threonine protein kinase,can play an important role in the plant stress signal transduction pathway through phosphorylation.We analyzed the expression pattern of SnRK2.4 under abiotic stresses,aiming to reveal its role in the regulation of adversity.The SnRK2.4 gene of high-quality cold-season turfgrass Poa pratensis L. was cloned using RT-PCR,the SnRK2.4 gene contained an ORF of 1 092 bp encoding a 363-amino acid protein,and its molecular characteristics were analyzed by bioinformatics.In addition,the expression pattern of this gene in different tissue parts under different abiotic stresses was observed using qPCR.The results showed that the Poa pratensis L.SnRK2.4 gene belonged to the SRK/SAPK superfamily,contained typical STKc_SnRK2 domain,tyrosine kinase phosphorylation site,casein kinase Ⅱ phosphorylation site and serine/threonine-protein kinase activity site,which had the highest homology with Brachypodium distachyon.The qPCR results demonstrated that the Poa pratensis L.SnRK2.4 gene was tissue-specific,highly expressed in panicles,and had no significant differential expression in roots,stems and leaves.Moreover,the Poa pratensis L.SnRK2.4 gene could respond positively to abiotic stresses such as drought,salt,low nitrogen,low phosphorus,ABA and BR.

Key words: Kentucky bluegrass, Protein kinase, SnRK2.4, Gene cloning, Abiotic stress

金一锋, 高岩松, 王琦, 王萌萌, 赵迪, 熊毅, 陈阳. 草地早熟禾蛋白激酶SnRK2.4 基因克隆及非生物胁迫响应分析[J]. 华北农学报, 2022, 37(5): 9-15. doi: 10.7668/hbnxb.20193082.

JIN Yifeng, GAO Yansong, WANG Qi, WANG Mengmeng, ZHAO Di, XIONG Yi, CHEN Yang. Cloning of SnRK2.4 Gene and It's Response to Abiotic Stress in Kentucky Bluegrass[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 9-15. doi: 10.7668/hbnxb.20193082.

http://www.hbnxb.net/CN/Y2022/V37/I5/9

图/表 8

表1 所需RT-PCR及qRT-PCR引物

Tab.1 The primers used for RT-PCR and qRT-PCR

引物名称 Primer name	序列(5'—3') Sequences(5'—3')	长度/bp Length	功能 Function
SnRK2.4-F	GACATCGGGTCGGGCAACTT	1 242	基因克隆
SnRK2.4-R	CCACTGAACTGAAATCCCCG
Q-SnRK2.4-F	TTGAAGACCCAGATGACCCC	155	qRT-PCR
Q-SnRK2.4-R	CATTGTGATTCTCCTCGCAG
Q-UBQ-F	AGAAGAAGACCTACACCAAG	217	内参基因
Q-UBQ-R	GGTTGTAAGGCGTAGGTGAG

图1 草地早熟禾SnRK2.4 基因PCR扩增产物的电泳

Fig.1 Electropherogram of PCR-amplified products of SnRK2.4

图2 草地早熟禾SnRK2.4氨基酸的保守结构域分析

Fig.2 Conserved domain analysis of SnRK2.4 in kentucky bluegrass

图3 草地早熟禾与其他植物SnRK2.4的系统进化树

Fig.3 Phylogenetic tree of SnRK2.4 in kentucky bluegrass and other plants

图4 草地早熟禾与二穗短柄草、长穗偃麦草和大麦SnRK2.4同源蛋白的多序列比对 Ⅰ.蛋白激酶ATP结合信号区;Ⅱ.丝氨酸/苏氨酸蛋白激酶活性位点;Ⅲ.富含谷氨酸的区域;-.cAMP 和 cGMP 依赖性蛋白激酶磷酸化位点;=.N-肉豆蔻酰化位点;#.酪氨酸激酶磷酸化位点;^.酪蛋白激酶Ⅱ磷酸化位点。

Fig.4 Multiple sequence alignment of SnRK2.4 homologous proteins of kentucky bluegrass, Brachypodium,Hordeum vulgare and Thinopyrum elongatum Ⅰ.Protein kinase ATP binding signal domain;Ⅱ.Serine/threonine protein kinase active site;Ⅲ.Glutamate-rich region;-.cAMP and cGMP-dependent protein kinase phosphorylation site;=.N-Myristoylation site;#.Tyrosine kinase phosphorylation site;^.Casein kinase Ⅱphosphorylation site.

图5 草地早熟禾SnRK2.4蛋白质的二级、三级结构

Fig.5 Secondary and tertiary structure of SnRK2.4 protein in kentucky bluegrass

图6 草地早熟禾SnRK2.4基因的组织特异性分析不同小写字母表示组织部位中基因表达差异显著 (P<0.05)。

Fig.6 Expression level analysis of SnRK2.4 in kentucky bluegrass under tissue Different lowercase letters indicate significant differences among tissue(P<0.05).

图7 草地早熟禾SnRK2.4基因在非生物胁迫下的表达模式不同小写字母表示干旱、盐、氮、磷、ABA、BR 处理过程中目的基因表达差异显著 (P<0.05)。

Fig.7 Expression pattern of SnRK2.4 gene in kentucky bluegrass under abiotic stress Different lowercase letters indicate significant differences during drought,salt,nitrogen,phosphorus,ABA and BR stress(P<0.05).

参考文献 23

[1]	Sun X Y, Zheng Q J, Xiong L B, Xie F C, Li X, Li Y, Zhang L, Saud S, Guo Z X, Yan Y, Wu H F, Liu Q Q, Cui G W, Chen Y J. Nitrogen assimilation and gene regulation of two Kentucky bluegrass cultivars differing in response to nitrate supply[J]. Scientia Horticulturae, 2021, 288:110315.doi: 10.1016/J.SCIENTA.2021.110315. doi: 10.1016/J.SCIENTA.2021.110315 URL
[2]	Coello P, Hey S J, Halford N G. The sucrose non-fermenting-1-related(SnRK)family of protein kinases:Potential for manipulation to improve stress tolerance and increase yield[J]. Journal of Experimental Botany, 2011, 62(3):883-893.doi: 10.1093/jxb/erq331. doi: 10.1093/jxb/erq331 URL
[3]	袁敏, 葛伟娜, 王莉, 张岚, 张家琦. 拟南芥蛋白激酶SnRK1.1对转录因子FD的磷酸化分析[J]. 华北农学报, 2017, 32(4):37-41.doi: 10.7668/hbnxb.2017.04.006. doi: 10.7668/hbnxb.2017.04.006
	Yuan M, Ge W N, Wang L, Zhang L, Zhang J Q. Protein kinase SnRK1.1 phosphorylates transcriptional factor FD in Arabidopsis thaliana[J]. Acta Agriculturae Boreali-Sinica, 2017, 32(4):37-41.
[4]	Lu K, Zhang Y D, Zhao C F, Zhou L H, Zhao Q Y, Chen T, Wang C L. The Arabidopsis kinase-associated protein phosphatase KAPP,interacting with protein kinases SnRK2.2/2.3/2.6,negatively regulates abscisic acid signaling[J]. Plant Molecular Biology, 2020, 102(1/2):199-212.doi: 10.1007/s11103-019-00941-8. doi: 10.1007/s11103-019-00941-8 URL
[5]	Ma X, Li Q H, Yu Y N, Qiao Y M, Haq S U, Gong Z H. The CBL CIPK pathway in plant response to stress signals[J]. International Journal of Molecular Sciences, 2020, 21(16):5668.doi: 10.3390/ijms21165668. doi: 10.3390/ijms21165668 URL
[6]	Diédhiou C J, Popova O V, Dietz K J, Golldack D. The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice[J]. BMC Plant Biology, 2008, 8:49.doi: 10.1186/1471-2229-8-49. doi: 10.1186/1471-2229-8-49 pmid: 18442365
[7]	Krzywińska E, Kulik A, Bucholc M, Fernandez M A, Rodriguez P L, Dobrowolska G. Protein phosphatase type 2C PP2CA together with ABI1 inhibits SnRK2.4 activity and regulates plant responses to salinity[J]. Plant Signaling & Behavior, 2016, 11(12):e1253647.doi: 10.1080/15592324.2016.1253647. doi: 10.1080/15592324.2016.1253647
[8]	Szymańska K P, Polkowska-Kowalczyk L, Lichocka M, Maszkowska J, Dobrowolska G. SNF1-related protein kinases SnRK2.4 and SnRK2.10 modulate ROS homeostasis in plant response to salt stress[J]. International Journal of Molecular Sciences, 2019, 20(1):143-143.doi: 10.3390/ijms20010143. doi: 10.3390/ijms20010143 URL
[9]	李荣, 冯月娟, 王舰, 王芳. 马铃薯StSnRK2.4基因的序列分析及其在非生物胁迫下的表达分析[J]. 分子植物育种, 2021, 19(22):7327-7336.doi: 10.13271/j.mpb.019.007327. doi: 10.13271/j.mpb.019.007327
	Li R, Feng Y J, Wang J, Wang F. Sequence analysis and expression analysis under abiotic stress of potato StSnRK2.4 gene[J]. Molecular Plant Breeding, 2021, 19(22):7327-7336.
[10]	Wang L Z, Hu W, Sun J T, Liang X Y, Yang X Y, Wei S Y, Wang X T, Zhou Y, Xiao Q, Yang G X, He G Y. Genome-wide analysis of SnRK gene family in Brachypodium distachyon and functional characterization of BdSnRK2.9[J]. Plant Science, 2015, 237:33-45.doi: 10.1016/j.plantsci.2015.05.008. doi: 10.1016/j.plantsci.2015.05.008 URL
[11]	Chen Z W, Zhou L H, Jiang P P, Lu R J, Halford N G, Liu C H. Genome-wide identification of sucrose nonfermenting-1-related protein kinase(SnRK)genes in barley and RNA-seq analyses of their expression in response to abscisic acid treatment[J]. BMC Genomics, 2021, 22(1):300.doi: 10.1186/s12864-021-07601-6. doi: 10.1186/s12864-021-07601-6 URL
[12]	Zhong R L, Wang Y X, Gai R N, Xi D D, Mao C J, Ming F. Rice SnRK protein kinase OsSAPK8 acts as a positive regulator in abiotic stress responses[J]. Plant Science, 2020, 292:110373.doi: 10.1016/j.plantsci.2019.110373. doi: 10.1016/j.plantsci.2019.110373 URL
[13]	Kobayashi Y, Yamamoto S, Minami H, Kagaya Y, Hattori T. Differential activation of the rice sucrose nonfermenting1-related protein Kinase2 family by hyperosmotic stress and abscisic acid[J]. The Plant Cell, 2004, 16(5):1163-1177.doi: 10.1105/tpc.019943. doi: 10.1105/tpc.019943 URL
[14]	Saito S, Hamamoto S, Moriya K, Matsuura A, Sato Y, Muto J, Noguchi H, Yamauchi S, Tozawa Y, Ueda M, Hashimoto K, Köster P, Dong Q Y, Held K, Kudla J, Utsumi T, Uozumi N. N-myristoylation and S-acylation are common modifications of Ca²⁺-regulated Arabidopsis kinases and are required foractivation of the SLAC1 anion channel[J]. New Phytologist, 2018, 218(4):1504-1521.doi: 10.1111/nph.15053. doi: 10.1111/nph.15053 URL
[15]	Nugent F S, Niehaus J L, Kauer J A. PKG and PKA signaling in LTP at GABAergic synapses[J]. Neuropsychopharmacology, 2009, 34(7):1829-1842.doi: 10.1038/npp.2009.5. doi: 10.1038/npp.2009.5 pmid: 19194373
[16]	McLoughlin F, Galvan-Ampudia C S, Julkowska M M, Caarls L, van der Does D, Laurière C, Munnik T, Haring M A, Testerink C. The Snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress[J]. The Plant Journal, 2012, 72(3):436-449.doi: 10.1111/j.1365-313X.2012.05089.x. doi: 10.1111/j.1365-313X.2012.05089.x pmid: 22738204
[17]	Lou D J, Chen Z, Yu D Q, Yang X Y. SAPK2 contributes to rice yield by modulating nitrogen metabolic processes under reproductive stage drought stress[J]. Rice, 2020, 13(1):35.doi: 10.1186/s12284-020-00395-3. doi: 10.1186/s12284-020-00395-3 pmid: 32514747
[18]	Su H, Wang T, Ju C F, Deng J P, Zhang T Q, Li M J, Tian H, Wang C. Abscisic acid signaling negatively regulates nitrate uptake via phosphorylation of NRT1.1 by SnRK2s in Arabidopsis[J]. Journal of Integrative Plant Biology, 2021, 63(3):597-610.doi: 10.1111/jipb.13057. doi: 10.1111/jipb.13057 URL
[19]	Joo J, Lee Y H, Song S I. OsbZIP42 is a positive regulator of ABA signaling and confers drought tolerance to rice[J]. Planta, 2019, 249(5):1521-1533.doi: 10.1007/s00425-019-03104-7. doi: 10.1007/s00425-019-03104-7 pmid: 30712129
[20]	Mao X G, Zhang H Y, Tian S J, Chang X P, Jing R L. 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, 2009, 61(3):683-696.doi: 10.1093/jxb/erp331. doi: 10.1093/jxb/erp331 URL
[21]	Miao L L, Li Y Y, Zhang H J, Zhang H J, Liu X L, Wang J Y, Chang X P, Mao X G, Jing R L. TaSnRK2.4 is a vital regulator in control of thousand-kernel weight and response to abiotic stress in wheat[J]. Journal of Integrative Agriculture, 2021, 20(1):46-54.doi: 10.1016/S2095-3119(19)62830-3. doi: 10.1016/S2095-3119(19)62830-3
[22]	Wang C T, Abbas F, Zhou Y W, Ke Y G, Li X Y, Yue Y C, Yu Y Y, Yu R C, Fan Y P. Genome-wide identification and expression pattern of SnRK gene family under several hormone treatments and its role in floral scent emission in Hedychium coronarium[J]. Peerj, 2021, 9,e10883.doi: 10.7717/peerj.10883. doi: 10.7717/peerj.10883 URL
[23]	Huai J L, Wang M, He J G, Zheng J, Dong Z G, Lü H K, Zhao J F, Wang G Y. Cloning and characterization of the SnRK2 gene family from Zea mays[J]. Plant Cell Reports, 2008, 27(12):1861-1868.doi: 10.1007/s00299-008-0608-8. doi: 10.1007/s00299-008-0608-8 URL

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草地早熟禾蛋白激酶SnRK2.4 基因克隆及非生物胁迫响应分析

Cloning of SnRK2.4 Gene and It's Response to Abiotic Stress in Kentucky Bluegrass

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草地早熟禾蛋白激酶SnRK2.4 基因克隆及非生物胁迫响应分析

Cloning of SnRK2.4 Gene and It's Response to Abiotic Stress in Kentucky Bluegrass

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