甘蔗CIPK基因的同源克隆与表达

作物学报 ›› 2015, Vol. 41 ›› Issue (03): 499-506.

甘蔗CIPK基因的同源克隆与表达

黄珑，苏炜华，张玉叶，黄宁，凌辉，肖新换，阙友雄，陈如凯

福建农林大学 / 农业部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗产业技术研发中心, 福建福州 350002

收稿日期:2014-07-14 修回日期:2014-12-09 出版日期:2015-03-12 网络出版日期:2014-12-30
通讯作者: 阙友雄, E-mail: queyouxiong@126.com; 陈如凯, fafu948@126.com
基金资助:
本研究由国家自然科学基金项目(31340060), 福建省高等学校新世纪优秀人才支持计划项目(JA14095)和国家现代农业产业技术体系建设专项(CARS-20)资助。

Cloning and Expression Analysis of CIPK Gene in Sugarcane

HUANG Long,SU Wei-Hua,ZHANG Yu-Ye,HUANG Ning,LING Hui,XIAO Xin-Huan,QUE You-Xiong*,CHEN Ru-Kai*

Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University / Sugarcane Research & Development Center, China Agricultural Technology System, Fuzhou 350002, China

Received:2014-07-14 Revised:2014-12-09 Published:2015-03-12 Published online:2014-12-30
Contact: 阙友雄, E-mail: queyouxiong@126.com; 陈如凯, fafu948@126.com

摘要/Abstract

摘要：

CIPK (calcineurin B-like-interacting protein kinase)是植物特有一类的丝氨酸/苏氨酸蛋白激酶，该蛋白在植物响应逆境胁迫中发挥着重要的作用，尤其与非生物逆境胁迫(干旱、高盐、ABA等)的信号传导密切相关。根据玉米CIPK15基因(EU957447.1, 2247 bp)核酸序列保守区域设计1对同源克隆PCR引物，以甘蔗品种崖城05-179的cDNA为模板，通过RT-PCR扩增得到甘蔗CIPK基因的一条全长cDNA序列(GenBank登录号为KM114052)。序列分析结果表明，甘蔗ScCIPK基因全长1782 bp，具有完整的开放阅读框(ORF, 91~1631 bp)，编码513个氨基酸，该基因具有CIPK基因的2个特征结构域(Kc_-like superfamily和AMPKA_-C_-like superfamily)。生物信息学分析显示该基因编码的蛋白定位于内质网，为可溶性蛋白，不存在信号肽，二级结构元件多为α-螺旋，含有多个保守功能域，主要参与中间代谢。实时定量PCR表达分析表明, 该基因表达具有组织特异性，虽在甘蔗各组织中均有表达，但在芽中的表达量最高。该基因在PEG、NaCl、ABA、SA和MeJA的胁迫诱导过程中，受ABA胁迫后表达量最高，约为对照的5.3倍，推测该基因的表达与甘蔗抗干旱和抗渗透胁迫有关。

关键词: 甘蔗, CIPK 基因, 同源克隆, 生物信息学, 实时定量PCR

Abstract:

CIPK (calcineurin B-like-interacting protein kinase) is a plant specific class of serine / threonine protein kinases, which plays an important role in plant response to stress, especially relates with the signal transduction for biotic stresses (drought, high salt, ABA). According to the primers designed on the conserved domain of CIPK15 gene from Zea mays, a full-length cDNA sequence of serine/threonine kinase gene termed as ScCIPK was cloned by RT-PCR method from sugarcane (Saccharum Complex). The sequence analysis showed that ScCIPK had a length of 1782 bp containing the open reading frame (ORF, 91–1631 bp), which encoded 513 amino acids residues with two conserved domains (Kc-like superfamily and AMPKA-C-like superfamily). The characters predicted based on the bioinformatics analysis revealed that the ScCIPK gene of sugarcane was a soluble acidic protein, which has two conserved functional domains with the main function for central_intermediary_metabolism, and its protein was located in endoplasmic reticulum (membrane). The mainly secondary structure element was α-helix. Real-time quantitative PCR(RT-qPCR) analysis revealed that the expression of ScCIPK was higher in bud than in other tissues, meanwhile the inducible expression level of ScCIPK was most significantly up-regulated under the ABA stress, 5.3 times higher than that of control, which suggested that ScCIPK most probably involves in sugarcane resistance to drought and osmotic stresses. The results in this study could provide a basis of cloning and functional identification of other members of ScCIPK in sugarcane and promote the use of ScCIPK gene in sugarcane genetic engineering.

Key words: Sugarcane, CIPK, homology cloning, Bioinformatics, Real-time PCR

黄珑，苏炜华，张玉叶，黄宁，凌辉，肖新换，阙友雄，陈如凯. 甘蔗CIPK基因的同源克隆与表达[J]. 作物学报, 2015, 41(03): 499-506.

HUANG Long,SU Wei-Hua,ZHANG Yu-Ye,HUANG Ning,LING Hui,XIAO Xin-Huan,QUE You-Xiong*,CHEN Ru-Kai*. Cloning and Expression Analysis of CIPK Gene in Sugarcane[J]. Acta Agron Sin, 2015, 41(03): 499-506.

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参考文献

[1]Zhu J K. Salt and drought strsee signal transdutiong in plants. Annu Rev Plant Biol, 2002, 53: 247–273

[2]Zhao J F, Sun Z F. Cloning and characterrization of a novel CBL-interracting protein kinase from maize. Plant Mol Biol, 2009, 69: 661–674

[3]王琦, 王伟, 申腾飞, 薛瑞丽, 邰付菊. 玉米中3个CIPK同源基因在干旱和低温胁迫下的表达分析. 华中农业大学学报, 2011, 30: 545–551

Wang Q, Wang W, Sheng T F, Xue R L, Tai F J. Three CIPK of homologous genes in maize under drought and low temperature stress analysis of the expression. J Huazhong Agric Univ, 2011, 30: 545–551 (in Chinese with English abstract)

[4]Albrecht V, Ritz O, Linder S, Harter K, Kudla J. The NAF domain defines a novel protein-protein interaction module in Ca2+-regulated kinases. EMBO J, 2001, 20: 1051–1063

[5]Lee S C, Lan W Z, Kim B G, Lin L, Cheong Y H, Pandey G K, Lu G H, Buchanan B B, Luan S. A protein phosphorylation/dephosphorylation network regulates a plant potassium channe. Proc Natl Acad Sci USA, 2007, 104: 15959–15964

[6]Sánchez-Barrena M J, Fujii H, Angulo I, Martínez-Ripoll M, Zhu J K, Albert A. The structure of the C-terminal domain of the protein kinase AtSOS2 bound to the calcium sensor AtSOS3. Mol Cell, 2007, 26: 427–435

[7]郭晋隆, 李国印, 苏亚春, 王恒波, 阙友雄, 徐景升, 许莉萍. 甘蔗R2R3-MYB类似基因Sc2RMyb1的克隆及表达特性分析. 农业生物技术学报, 2012, 20: 1009–1017

Guo J L, Li G Y, Su Y S, Wang H B, Que Y X, Xu J S, Xu L P. Sugarcane R2R3 MYB-similar gene cloning and expression of Sc2RMyb1 characteristics analysis. J Agric Biotechnol, 2012, 20: 1009–1017 (in Chinese with English abstract)

[8]Ye C Y, Xia X L, Yin W L. Evolutionary analysis of CBL-Interacting protein kinase gene family in plants. Plant Growth Regul, 2013, 71: 49–56

[9]Yu Y H, Xia X L, Yin W L, Zhang H C. Comparative genomic analysis CIPK gene family in Arabidopsis and Populus. Plant Growth Regul, 2007, 52: 101-110

[10]Zhang H F, Yang B, Liu W Z, Li H W, Wang L, Wang B Y, Deng M, Liang W W, Deyholos K M, Jiang Y Q. Identification and characterication of CBL and CIPK gene families in canola (Brassica napus). BMC Plant Biol, 2014, 14: 8–32

[11]Li L B, Zhang Y R, Liu K. C, Ni Z F, Fang Z J, Sun Q X, Gao J W. Identification and bioinformatics analysis of SnRK2 and CIPK family genes in sorghum. Agric Sci China, 2010, 9: 19–30

[12]Priji P J, Hemaprabha G.. Sugarcane specific drought responsive candidate genes belonging to ABA dependent pathway identified from basic species clones of Saccharum sp. and Erianthus sp. Sugar Tech, 2014, DOI 10.1007/s12355-014-0313-6

[13]邓小敏. 小麦CBL基因和CIPK基因的克隆及在非生物胁迫种的功能研究. 华中科技大学博士学位论文, 湖北武汉, 2013

Deng X M. Study on Wheat CBL and CIPK Genes Cloning and the Function under Abiotic Stress. PhD Dissertation of Huazhong University of Science and Technology, Wuhan, China, 2013 (in Chinese with English abstract)

[14]Chen L, Wang Q Q, Zhou L, Ren F, Li D D, Li X B. Aradidopsis CBL-interacting protein kinase(CIPK6) is involved in plant response to salt/osmotic stress and ABA. Mol Biol Rep, 2013, 40: 4759–4767

[15]边鸣镝, 吴忠义, 赵久然, 王永勤, 张秀海, 黄从林, 曹鸣庆. 非生物胁迫诱导的玉米蛋白激酶基因ZmCIPK1的cDNA克隆和表达分析. 农业生物技术学报, 2008, 16: 965–970

Bian M D, Wu Z Y, Zhao J R, Wang Y Q, Zhang X H, Huang C L, Cao M Q. The corn protein kinase gene induced by abiotic stress ZmCIPK1 cDNA cloning and expression analysis. J Agric Biotechnol, 2008, 16: 965–970 (in Chinese with English abstract)

[16]阙友雄, 许莉萍, 徐景升, 张积森, 张木清, 陈如凯. 甘蔗基因表达定量PCR分析中内参基因的选择. 热带作物学报, 2009, 30: 274–278

Que Y X, Xu L M, Xu J S, Zhang J S, Zhang M Q, Chen R K. Sugarcane quantitative PCR analysis of gene expression in the choice of internal genes. Chin J Trop Crops, 2009, 30: 274–278 (in Chinese with English abstract)

[17]Livak K J, Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the 2−ΔΔCT method. Methods, 2001, 25: 402–408

[18]张志勇, 雷朝云, 蒙秋伊, 卢加举. 甘蔗抗逆基因工程育种研究进展. 核农学报, 2012, 26: 471–477

Zhang Z Y, Lei Z Y, Meng Q Y, Lu J Y. Sugarcane art research progress of genetic engineering breeding. J Nucl Agric Sci, 2012, 26: 471–477 (in Chinese with English abstract)

[19]Quintero F J, Ohta M, Shi H, Zhu J K, Pardo J M. Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostassis. Proc Acad Natl Sci USA, 2002, 99: 9061–9066

[20]D. Angelo C, Weinl S, Batistic O, Pandey G K, Cheong Y H, Schultke S, Albrecht V, Ehlert B, Schulz B, Harter K, Luan S, Bock R, Kudla J. Altrnative complex formation of the Ca+-regulated protein kinase CIPK1 controls abscisic acid dependent and independent stress responses in Arabidopsis. Plant J, 2006, 48: 857–872

[21]Yu Q Y, An L J, Li W L. The CBL-CIPK network mediates different signaling pathways in plants. Plant Cell Rep, 2014, 33: 203–214

[22]李率帅, 阚国仕, 魏建华, 李瑞芬. 野大麦CIPK基因的亚细胞定位. 辽宁农业学报, 2011, (4): 1–5

Li S S, Kan G S, Wei J H, Li R F. The subcellular localization of wild barley CIPK gene. J Liaoning Agric, 2011, (4): 1–5 (in Chinese with English abstract)

[23]赵晋锋, 余爱丽, 王寒玉, 杜艳伟, 王高鸿, 常海霞. 非生物逆境胁迫下ZmCIPK10基因表达分析. 生物技术进展, 2011, 1(2): 130–134

Zhao J F, Yu A L, Wang H Y, Du Y W, Wang G H, Chang H X. Abiotic ZmCIPK10 gene expression under adversity stress analysis. Biotechnol Adv, 2011, 1(2): 130–134 (in Chinese with English abstract)

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