小麦类钙调素<i>TaCML8-A</i>基因的克隆和表达分析

doi:10.7668/hbnxb.20192627

摘要/Abstract

摘要：

为挖掘小麦抗逆基因,研究类钙调素基因在植物抗逆反应的分子机制,利用电子克隆结合RT-PCR的方法克隆小麦类钙调素基因TaCML8-A。生物信息学分析显示,该基因的CDS区全长为519 bp,编码172个氨基酸,包含PTZ00184和FRQ1超家族,含有4个EF-hand结构域,其中包含4个钙离子结合位点;编码蛋白质分子质量为18.31 ku,等电点为4.54,属于酸性蛋白质。亚细胞定位结果显示,TaCML8-A蛋白质在细胞核和细胞膜中均有分布。核苷酸序列比对发现,TaCML8-A与水稻OsCML14亲缘关系最近,相似性为79.17%。qRT-PCR结果显示,在盐、渗透和冷胁迫处理中,TaCML8-A基因在小麦地上部分和根中的表达均上升,表明植物可能通过提高TaCML8-A基因的表达来响应盐、渗透和冷胁迫;热激时TaCML8-A基因在根和地上部分分别受到诱导和抑制,从而发挥不同的功能。从小麦中成功克隆出类钙调素基因TaCML8-A,并进行表达分析,初步推测该基因可能参与调控植物的非生物胁迫,其结果可为后续研究其生物学功能提供基础理论支撑。

关键词: 小麦, 类钙调素, EF-hand结构域, 基因克隆, 基因表达

Abstract:

In order to explore stress resistance genes in wheat and study the molecular mechanism of calmodulin-like genes in plant stress resistance,a calmodulin-like gene TaCML8-A was cloned from wheat by electronic cloning combined with RT-PCR.Bioinformatics analysis showed that the open reading frame length of the gene was 519 bp,encoding 172 amino acid sequences,including PTZ00184 and FRQ1 superfamily,four EF-hand domains,including four calcium binding sites.The molecular weight of the encoded protein was 18.31 ku and the isoelectric point was 4.54.It belonged to acidic protein.Subcellular localization showed that TaCML8-A protein was distributed in the nucleus and cell membrane.Nucleotide sequence alignment showed that TaCML8-A had the closest genetic relationship with rice OsCML14,with a similarity of 79.17%.qRT-PCR results showed that the expression of TaCML8-A gene in shoot and root of wheat increased under salt,osmotic and cold stress,indicating that plants might respond to thoese stresses by increasing the expression of TaCML8-A.During heat shock,TaCML8-A gene was induced and inhibited in root and shoot,respectively,so as to play different functions.The calmodulin-like gene TaCML8-A was successfully cloned from wheat and analyzed for expression.It is preliminarily speculated that the gene might be involved in regulating abiotic stress of plants,so as to provide basic theoretical support for the follow-up study of its biological function.

Key words: Triticum aestivum, Calmodulin-like protein, EF-hand domain, Gene cloning, Gene expression

贾伟哲, 焦博, 王娇, 陈文烨, 杨帆, 刘永伟, 董福双, 赵立群, 周硕. 小麦类钙调素TaCML8-A基因的克隆和表达分析[J]. 华北农学报, 2022, 37(1): 1-8. doi: 10.7668/hbnxb.20192627.

JIA Weizhe, JIAO Bo, WANG Jiao, CHEN Wenye, YANG Fan, LIU Yongwei, DONG Fushuang, ZHAO Liqun, ZHOU Shuo. Cloning and Expression Analysis of Calmodulin-like Protein TaCML8-A Gene in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 1-8. doi: 10.7668/hbnxb.20192627.

http://www.hbnxb.net/CN/Y2022/V37/I1/1

图/表 9

表1 引物序列信息

Tab.1 Primer sequence information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Application
TaCML8-A	F:ATGACGAACCCATCGCCGG	RT-PCR
	R:TTAGGCGGCGGGGACTCCG
TaCML8-A-Q	F:TTCGAGGAGCTGCGGACCAT	qPCR
	R:CCCGGCCGGTCATCATCTCA
18S rRNA	F:GATCCATTGGAGGGCAAGTC	qPCR
	R:GATGCTTGCTTTGAGCACTC
TaCML8-A-EcoR Ⅰ/Hind Ⅲ	F:CGGAATTCATGACGAACCCATCGCCGG	载体构建
	R:CCCAAGCTTTTAGGCGGCGGGGACTCCG

表2 生物信息学分析软件及内容

Tab.2 Bioinformatics analysis software and content

分析内容 Analytical contents	软件名称 Software brand	网址 Web sites
基因序列的ORF预测	ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/
ORF prediction of gene sequences
蛋白分子量及等电点分析	ExPASy ProtParam	https://web.expasy.org/protparam/
Protein molecular weight and isoelectric point analysis
蛋白的保守结构域分析	ProSite	http://prosite.expasy.org/
Conservative domain analysis of proteins
多重序列比对及相似度分析	Clustal Omega	https://www.ebi.ac.uk/Tools/msa/clustalo/
Multi-sequence comparison and similarity analysis
多重序列比对结果的输出着色	BoxShade	https://embnet.vital-it.ch/software/ BOX_form.html/
Multi-sequence comparison of the output of the results
构建系统进化树	MEGA 6.0	http://www.megasoftware.net/
Construction of phylogenetic tree

图1 小麦TaCML8-A基因克隆
D2000.D2000 DNA Marker;TaCML8-A.TaCML8-A基因的扩增片段。

Fig.1 The cloning of TaCML8-A gene in wheat
D2000.D2000 DNA Marker;TaCML8-A.Gene amplification of TaCML8-A.

图2 预测的TaCML8-A蛋白保守结构域

Fig.2 Predicted conserved domain of TaCML8-A protein

图3 5种植物的TaCML8-A蛋白多序列比对
横线为EF手型结构域;虚线为Ca²⁺结合结构域。

Fig.3 Multiple sequence alignment of TaCML8-A protein in 5 plants
The horizontal line is EF-hand domain;The dotted line is the Ca²⁺ binding domain.

图4 8种植物的CaM/CML蛋白系统进化树分析

Fig.4 Phylogenetic tree of CaM/CML proteins in 8 plants

图5 TaCML8-A蛋白在烟草叶肉细胞的亚细胞定位

Fig.5 Subcellular localization of TaCML8-A protein in tobacco mesophyll cells

图6 TaCML8-A基因的组织表达特异性
1.幼嫩根;2.幼嫩的地上部分;3.成熟根;4.成熟茎;5.成熟叶;6.成熟花;7.种子。不同小写字母表示差异显著(P <0.05)。图7同。

Fig.6 Tissue expression specificity of TaCML8-A gene
1.Young root;2.Young shoot;3.Mature root;4.Mature stem;5.Mature leaf;6.Mature flower;7.Seed.Different small letters indicate significant difference(P <0.05).The same as Fig.7.

图7 TaCML8-A基因在各种胁迫下的相对表达

Fig.7 Relative expression of TaCML8-A gene in abiotic stresses

参考文献 28

[1]	郑仲仲,沈金秋,潘伟槐,潘建伟.植物钙感受器及其介导的逆境信号途径[J].遗传,2013,35(7):875-884.doi:10.3724/SP.J.1005.2013.00875.
	Zheng Z Z,Shen J Q,Pan W H,Pan J W.Calcium sensors and their stress signaling pathways in plants[J].Hereditas,2013,35(7):875-884.
[2]	Dodd A N,Kudla J,Sanders D.The language of calcium signaling[J].Annual Review of Plant Biology,2010,61:593-620.doi:10.1146/annurev-arplant-070109-104628.
[3]	Reddy A S N,Ali G S,Celesnik H,Day I S.Coping with stresses:Roles of calcium-and calcium/calmodulin-regulated gene expression[J].The Plant Cell,2011,23(6):2010-2032.doi:10.1105/tpc.111.084988.
[4]	McCormack E,Braam J.Calmodulins and related potential calcium sensors of Arabidopsis[J].The New Phytologist,2003,159(3):585-598.doi:10.1046/j.1469-8137.2003.00845.x.
[5]	Boonburapong B,Buaboocha T.Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins[J].BMC Plant Biology,2007,7:4.doi:10.1186/1471-2229-7-4.
[6]	霍建飞,刘春艳,郝永娟,王勇,王万立.植物中重要的信号分子——CaM[J].天津农业科学,2010,16(3):30-35.doi:10.3969/j.issn.1006-6500.2010.03.010.
	Huo J F,Liu C Y,Hao Y J,Wang Y,Wang W L.Important signal molecular in plants—CaM[J].Tianjin Agricultural Sciences,2010,16(3):30-35.
[7]	McCormack E,Tsai Y C,Braam J.Handling calcium signaling:Arabidopsis CaMs and CMLs[J].Trends in Plant Science,2005,10(8):383-389.doi:10.1016/j.tplants.2005.07.001.
[8]	Nelissen H,Clarke J H,De Block M,De Block S,Vanderhaeghen R,Zielinski R E,Dyer T,Lust S,Inzé D,Van Lijsebettens M.DRL1,a homolog of the yeast TOT4/KTI12 protein,has a function in meristem activity and organ growth in plants[J].The Plant Cell,2003,15(3):639-654.doi:10.1105/tpc.007062.
[9]	Senapati D, Kushwaha R, Dutta S, Maurya J P, Biswas S, Gangappa S N, Chattopadhyay S.COP1 regulates the stability of CAM7 to promote photomorphogenic growth[J].Plant Direct,2019,3(6):e00144.doi:10.1002/pld3.144.
[10]	Rao S S,El-Habbak M H,Havens W M,Singh A,Zheng D M,Vaughn L,Haudenshield J S,Hartman G L,Korban S S,Ghabrial S A.Overexpression of GmCaM4 in soybean enhances resistance to pathogens and tolerance to salt stress[J].Molecular Plant Pathology,2014,15(2):145-160.doi:10.1111/mpp.12075.
[11]	薛敏,王雪峰,王志林,李连国,赵鸿彬,王茅雁.沙冬青AmCaM1基因的克隆及其功能初步分析[J].植物遗传资源学报,2015,16(6):1315-1320.doi:10.13430/j.cnki.jpgr.2015.06.025.
	Xue M,Wang X F,Wang Z L,Li L G,Zhao H B,Wang M Y.Cloning and preliminary functional analysis of AmCaM1 from Ammopiptanthus mongolicus[J].Journal of Plant Genetic Resources,2015,16(6):1315-1320.
[12]	Chu M X,Li J J,Zhang J Y,Shen S F,Li C N,Gao Y J,Zhang S Q.AtCaM4 interacts with a Sec14-like protein,PATL1,to regulate freezing tolerance in Arabidopsis in a CBF-independent manner[J].Journal of Experimental Botany,2018,69(21):5241-5253.doi:10.1093/jxb/ery278.
[13]	Kim Y,Park S,Gilmour S J,Thomashow M F.Roles of CAMTA transcription factors and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis[J].The Plant Journal,2013,75(3):364-376.doi:10.1111/tpj.12205.
[14]	Liu H T,Li B,Shang Z L,Li X Z,Mu R L,Sun D Y,Zhou R G.Calmodulin is involved in heat shock signal transduction in wheat[J].Plant Physiology,2003,132(3):1186-1195.doi:10.1104/pp.102.018564.
[15]	Magnan F,Ranty B,Charpenteau M,Sotta B,Galaud J P,Aldon D.Mutations in AtCML9,a calmodulin-like protein from Arabidopsis thaliana,alter plant responses to abiotic stress and abscisic acid[J].The Plant Journal,2008,56(4):575-589.doi:10.1111/j.1365-313X.2008.03622.x.
[16]	Cho K M,Nguyen H T K,Kim S Y,Shin J S,Cho D H,Hong S B,Shin J S,Ok S H.CML10,a variant of calmodulin,modulates ascorbic acid synthesis[J].The New Phytologist,2016,209(2):664-678.doi:10.1111/nph.13612.
[17]	吴晓萌,李春龙,钱景美,郑昱,张伟.拟南芥钙调素类似蛋白CML20参与植物抗干旱研究[J].中国科技论文在线精品论文,2016,9(3):205-212.
	Wu X M,Li C L,Qian J M,Zheng Y,Zhang W.Plant drought response evokes CML20,a calmodulin-like protein of Arabidopsis thaliana[J].Sciencepaper Oline,2016,9(3):205-212.
[18]	Yamaguchi T,Aharon G S,Sottosanto J B,Blumwald E.Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca²+-and pH-dependent manner[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(44):16107-16112.doi:10.1073/pnas.0504437102.
[19]	Munir S,Khan M R G,Song J W,Munir S,Zhang Y Y,Ye Z B,Wang T T.Genome-wide identification,characterization and expression analysis of calmodulin-like(CML)proteins in tomato(Solanum lycopersicum)[J].Plant Physiology and Biochemistry,2016,102:167-179.doi:10.1016/j.plaphy.2016.02.020.
[20]	Leba L,Cheval C,Ortiz-Martín I,Ranty B,Beuzón C R,Galaud J,Aldon D.CML9,an Arabidopsis calmodulin-like protein,contributes to plant innate immunity through a flagellin-dependent signalling pathway[J].The Plant Journal,2012,71(6):976-989.doi:10.1111/j.1365-313X.2012.05045.x.
[21]	Vadassery J,Reichelt M,Hause B,Gershenzon J,Boland W,Mithöfer A.CML42-mediated calcium signaling coordinates responses to Spodoptera herbivory and abiotic stresses in Arabidopsis[J].Plant Physiology,2012,159(3):1159-1175.doi:10.1104/pp.112.198150.
[22]	Mohanta T K,Kumar P,Bae H H.Genomics and evolutionary aspect of calcium signaling event in calmodulin and calmodulin-like proteins in plants[J].BMC Plant Biology,2017,17(1):38.doi:10.1186/s12870-017-0989-3.
[23]	Yang T,Segal G,Abbo S,Feldman M,Fromm H.Characterization of the calmodulin gene family in wheat:Structure,chromosomal location,and evolutionary aspects[J].Molecular and General Genetics,1996,252(6):684-694.doi:10.1007/BF02173974.
[24]	Lu L,Rong W,Zhou R H,Huo N X,Zhang Z Y.TaCML36,a wheat calmodulin-like protein,positively participates in an immune response to Rhizoctonia cerealis[J].The Crop Journal,2019,7(5):608-618.doi:10.1016/j.cj.2019.02.001.
[25]	周硕,刘永伟,董福双,杨帆,赵和,柴建芳,吕孟雨,孙果忠,王海波.小麦类钙调素TaCML79基因的克隆和表达分析[J].华北农学报,2016,31(6):1-6.doi:10.7668/hbnxb.2016.06.001.
	Zhou S,Liu Y W,Dong F S,Yang F,Zhao H,Chai J F,Lǖ M Y,Sun G Z,Wang H B.Cloning and expression analysis of TaCML79 in wheat[J].Acta Agriculturae Boreali-Sinica,2016,31(6):1-6.
[26]	Delk N A,Johnson K A,Chowdhury N I,Braam J.CML24,regulated in expression by diverse stimuli,encodes a potential Ca²+ sensor that functions in responses to abscisic acid,daylength,and ion stress[J].Plant Physiology,2005,139(1):240-253.doi:10.1104/pp.105.062612.
[27]	Choi W G,Hilleary R,Swanson S J,Kim S H,Gilroy S.Rapid,long-distance electrical and calcium signaling in plants[J].Annual Review of Plant Biology,2016,67:287-307.doi:10.1146/annurev-arplant-043015-112130.
[28]	Yin X M,Huang L F,Zhang X,Wang M L,Xu G Y,Xia X J.OsCML4 improves drought tolerance through scavenging of reactive oxygen species in rice[J].Journal of Plant Biology,2015,58(1):68-73.doi:10.1007/s12374-014-0349-x.

选择文件类型/文献管理软件名称

选择包含的内容

小麦类钙调素TaCML8-A基因的克隆和表达分析

Cloning and Expression Analysis of Calmodulin-like Protein TaCML8-A Gene in Wheat

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价

[1]	许华杰, 卢莉莉, 汤寓涵, 赵大球, 孟家松, 陶俊. *芍药PlSPL1基因克隆与表达特性分析*[J]. 华北农学报, 2024, 39(2): 55-61.
[2]	王建伟, 李东晓, 王千一, 张明哲, 李瑞奇. 黄淮北部不同冬小麦品种(系)产量和氮素利用效率差异[J]. 华北农学报, 2024, 39(2): 143-152.
[3]	王慧珍, 张朝政, 黄一鸣, 黎耀心, 程子洋, 乐超银. *高粱抗病相关基因SbRPM1的克隆及表达分析*[J]. 华北农学报, 2024, 39(2): 153-160.
[4]	高泽仁, 潘鹏丞, 徐文文, 陈宝剑, 刘明君, 关志惠, 谢炳坤, 覃兆鲜. *陆川猪MyoD1基因克隆及组织表达分析*[J]. 华北农学报, 2024, 39(2): 168-173.
[5]	黄明, 姜沛沛, 张振旺, 吴金芝, 李友军. 干旱和品种对小麦灌浆期旗叶下午光合速率、关键酶活性和产量的影响[J]. 华北农学报, 2024, 39(2): 90-98.
[6]	胡莲, 刘益丽, 赵迪, 王泽宁, 江明锋. *麦洼牦牛MFSD4A基因克隆及组织表达分析*[J]. 华北农学报, 2024, 39(2): 174-183.
[7]	吴茵茵, 温思怡, 韩卓然, 孙敬锋. *半滑舌鳎突触体相关蛋白29基因(SNAP29)克隆及其组织分布和时序表达分析*[J]. 华北农学报, 2024, 39(2): 231-238.
[8]	崔永增, 韩江伟, 姚海坡, 姚艳荣, 吕丽华, 贾秀领. 有机肥替代氮肥对冬小麦氮素吸收利用的影响[J]. 华北农学报, 2024, 39(1): 135-142.
[9]	李凯丽, 耿明状, 王胜, 郝维浩, 卢杰, 陈璨, 司红起. *安农1687抗条锈病候选基因TraesCS2A01G070700鉴定与分析*[J]. 华北农学报, 2024, 39(1): 150-155.
[10]	赵欣雨, 康占海, 王羽, 李艳艳, 高维芳, 李星. 50个小麦品种抗叶锈基因鉴定[J]. 华北农学报, 2024, 39(1): 156-164.
[11]	贺彩霞, 李长忠, 保长虹, 王丽楠, 严青春, 金文杰, 赵娟, 王国杰, 简生龙, 王振吉, 陈艳霞. *花斑裸鲤MSTN-1基因克隆及表达特性分析*[J]. 华北农学报, 2024, 39(1): 228-238.
[12]	张自阳, 周谦, 王依, 王志伟, 朱启迪, 茹振钢, 刘明久. *小麦倒春寒响应基因TaJAZ6的克隆、表达模式及亚细胞定位分析*[J]. 华北农学报, 2024, 39(1): 27-36.
[13]	姚楚玄, 焦博, 秘彩莉, 杨帆, 张磊, 周硕, 柴建芳. 小麦品质育种2个早代选择参数的比较与优化[J]. 华北农学报, 2023, 38(S1): 34-38.
[14]	李义, 康占海, 董素芬, 王羽, 李艳艳, 赵欣雨, 李星. 75份国内外小麦品种抗叶锈性鉴定及基因分析[J]. 华北农学报, 2023, 38(S1): 362-370.
[15]	庄文锋, 翟军, 刘超, 李德正, 孔令让, 李安飞, 杨猛. 基本苗和行距对山农38小麦产量和光能利用的影响[J]. 华北农学报, 2023, 38(S1): 95-101.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

小麦类钙调素TaCML8-A基因的克隆和表达分析

Cloning and Expression Analysis of Calmodulin-like Protein TaCML8-A Gene in Wheat

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价