小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2015.08.01

摘要/Abstract

摘要： 【目的】克隆与逆境胁迫相关的基因，并对其序列特征、进化关系和表达特性进行分析，探讨该基因在小麦抗逆调控过程中的生物学功能，为进一步解析植物的抗逆机制提供候选基因和理论依据。【方法】以cDNA芯片数据获得的水分胁迫诱导上调表达基因EST序列为探针，对小麦EST数据库进行搜索，筛选与探针同源性在97％以上的EST序列，通过电子克隆结合RT-PCR获得该基因cDNA全长，采用生物信息学软件分析比较克隆基因的保守结构域及序列特征；采用MEGA6.0软件构建该基因的系统进化树；将测序正确的该基因片段通过EcoRⅠ和HindⅢ限制性内切酶酶切连接至原核表达载体pMAL-c2X，重组质粒转化大肠杆菌BL21，经终浓度为0.3 mmol·L^-1IPTG诱导1—5 h后，用SDS-PAGE分析融合蛋白的表达；采用实时荧光定量PCR（qRT-PCR）分析该基因在小麦不同组织间的表达差异及其在低温、干旱、高温和ABA处理下的表达模式。【结果】成功获得小麦cDNA全长序列，命名为TaC2DP1。该基因序列全长为1 356 bp，包含一个1 209 bp的开放阅读框（ORF），5′端非编码区50 bp，3′端非编码区97 bp，编码402个氨基酸，推导编码蛋白质的预测分子量为43.41 kD，等电点为4.30，属于酸性蛋白，BLAST分析表明，该蛋白含有一个与钙离子结合的结构域，称为C2结构域（C2-domain）。多序列比对及进化树分析表明，TaC2DP1与乌拉尔图小麦TuC2亲缘关系最近，二者具有高度的同源性，其编码的氨基酸一致性达到91%；蛋白质结构预测分析显示TaC2DP1无跨膜螺旋和双硫键，亚细胞定位于细胞质中；成功构建了该基因的原核表达载体pMAL-c2X-TaC2DP1，在IPTG诱导下得到90 kD左右的蛋白，与理论值一致。通过实时荧光定量PCR进行TaC2DP1表达分析，显示该基因在小麦的根、茎、叶、幼穗、未成熟籽粒、胚及胚乳中均有表达，其中在幼穗中表达量最高，在花后5 d籽粒中表达量最低。TaC2DP1可被植物激素ABA诱导而上调表达；干旱胁迫过程中，TaC2DP1受胁迫诱导呈稳定上调表达趋势；高温和低温胁迫过程中，TaC2DP1均在胁迫后的0.5 h迅速诱导上调表达，分别为对照的21和17倍。推测该基因可能参与小麦ABA 信号通路中对逆境胁迫的抗性反应。【结论】获得小麦TaC2DP1的全长序列，其编码蛋白含有与钙离子结合的C2结构域；在低温、干旱、高温和ABA逆境胁迫下，TaC2DP1属于依赖于ABA胁迫响应基因调控网络，可能在干旱、低温和热胁迫中发挥重要作用。

关键词: 小麦, C2结构域蛋白, 基因克隆, 表达分析

Abstract: 【Objective】The objective of this study is to clone the stress resistance-related gene, analyze its sequence features, evolutionary relationships and expression characteristics, investigate its biological function during the stress tolerance of wheat, and to provide candidate gene and a theoretical foundation for clarifying molecular mechanism of stress resistance.【Method】Using an up-regulated EST obtained by cDNA chip as a probe to search the wheat EST databases, filter out the ESTs sequences with the homology of 97% of the probe, a full-length cDNA sequence was cloned from wheat by in silico cloning and reverse transcription PCR (RT-PCR) method. The conserved domains and sequence features of the gene were analyzed by bioinformatics’ methods. A phylogenetic tree was constructed using the MEGA 6.0 software, and then the cloned geneORF was inserted into the expression vector pMAL-c2X by EcoR I and HindⅢ digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.3 mmol·L^-1 IPTG for 1-5 h. The expression of the fusion protein was detected by SDS-PAGE. The expression profiles of the cloned genein various tissues and in response to cold, drought, heat and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR).【Result】The full-length cDNA sequence designated as TaC2DP1 from wheat is 1 356 bp in length, contains a 1 209 bp open reading frame (ORF), with 50 bp in the 5' UTR and 97 bp in the 3' UTR. TaC2DP1was predicted to encode a 402 amino acid protein with a molecular mass of 43.41 kD and isoelectric point of 4.30, it belongs to the acidic protein. BLAST analysis revealed that the protein contains a C2-domain and was predicted to be a Ca²⁺ binding domain. Multiple sequence alignment and phylogenetic tree analysis showed that TaC2DP1 had the closest evolutionary relationship with a C2-domain protein in Triticum urartu with unknown function, and shares 91% identity in amino acids. Protein structure prediction showed that TaC2DP1 had not transmembrane helix and disulfide bondand might be localized in cytoplasm. The prokaryotic expression vector of TaC2DP1, pMAL-c2X-TaC2DP1,was successfully constructed. The expression of fusion protein was obtained by inducing with IPTG and its relative molecular weight was 90 kD, which was consistent with the theoretical value. Real time quantitative PCR (qRT-PCR) analysis revealed that TaC2DP1 constitutively expressed in all tested roots, stems, leaves, immature ears, immature seeds, embryo and endosperm, the expression level of TaC2DP1 in immature ears was the highest, while its expression level was very low in seeds at 5 days after anthesis (DAA). The expression profiling revealed that TaC2DP1 was induced by plant hormone ABA and the expression of TaC2DP1 was steadily up-regulated in any of the time point under drought stress. TaC2DP1 was rapidly up-regulated within 0.5 h of cold and heat stress treatments and the expression level was 21 and 17 times than those of control, respectively. These results revealed that TaC2DP1 might be involved in stress resistance-related response of ABA signaling pathways in wheat leaves.【Conclusion】A full-length cDNA of TaC2DP1 was isolated and the typical a Ca²⁺ binding domain was found in the deduced proteins. The expression of TaC2DP1 was all up-regulated under drought, high and low temperature and ABA treatments, showing that TaC2DP1 was involved in the ABA dependent stress-responding gene network.It was deduced that TaC2DP1 might play an important regulation role under stress in wheat.

Key words: wheat (Triticum aestivum), C2 domain protein, gene cloning, expression analysis

肖瑞霞，王新国，夏国军，李永春，牛洪斌，王翔，尹钧，任江萍. 小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析[J]. 中国农业科学, 2015, 48(8): 1463-1472.

XIAO Rui-xia, WANG Xin-guo, XIA Guo-jun, LI Yong-chun, NIU Hong-bin, WANG Xiang, YIN Jun, REN Jiang-ping. Cloning and Expression Analysis of A Stress-Related TaC2DP1 Gene from Wheat[J]. Scientia Agricultura Sinica, 2015, 48(8): 1463-1472.

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参考文献

[1] 刘诗航, 王彩香, 毛新国, 刘惠民, 李昂, 景蕊莲. 小麦蛋白磷酸酶 2A 调节亚基基因 TaBβ-1 的克隆及其在非生物胁迫下的表达特性. 中国农业科学, 2010, 43(11): 2197-2208.

Liu S H, Wang C X, Mao X G, Liu H M, Li A, Jing R L. Cloning of protein phosphatase 2A regulatory subunit gene TaBβ-1 and its expression patterns under abiotic stresses in wheat. Scientia Agricultura Sinica, 2010, 43(11): 2197-2208. (in Chinese)

[2] Tuteja N, Tiburcio A F, Fortes A M, Bartels D. Plant abiotic stress. Introduction to PSB special issue. Plant Signaling and Behavior, 2011, 6: 173-174.

[3] Qin F, Shinozaki K, Yamaguchi-Shinozaki K. Achievements and challenges in understanding plant abiotic stress responses and tolerance. Plant and Cell Physiology, 2011, 52: 1569-1582.

[4] Hirayama T, Shinozaki K. Research on plant abiotic stress responses in the post-genome era: Past, present and future. The Plant Journal, 2010, 61: 1041-1052.

[5] Jang J Y, Kim D G, Kim Y O, Kim J S, Kang H. An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Molecular Biology, 2004, 54: 713-725.

[6] Wegener C B, Jansen G.. Antioxidant capacity in cultivated and wild Solanum species: The effect of wound stress. Food and Function, 2010, 1: 209-218.

[7] Liu Y J, Zhang A N, Jia J F, Li A Z. Cloning of salt stress responsive cDNA from wheat and resistant analysis of differential fragment SR07 in transgenic tobacco. Journal of Genetics and Genomics, 2007, 34: 842-850.

[8] Andleeb S, Amin I, Bashir A, Briddon R W, Mansoor S. Transient expression of beta C1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions. Virology Journal, 2010, 7: 373.

[9] Chen W, Yao X, Cai K, Chen J. Silicon alleviates drought stress of rice plants by improving plant water status, photosynthesis and mineral nutrient absorption. Biological Trace Element Research, 2011, 142: 67-76.

[10] Corbalan-Garcia S, Gómez-Fernández J C. Signaling through C2 domains: More than one lipid target. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2014, 1838: 1536-1547.

[11] Coussens L, Parker P J, Rhee L, Yang-Feng T L, Chen E, Waterfield M D, Francke U, Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science, 1986, 233: 859-866.

[12] Kang C H, Moon B C, Park H C, Koo S C, Jeon J M, Cheong Y H, Chung W S, Lim C O, Kim J Y, Yoon B D, Lee S Y, Kim C Y. Rice OsERG3 encodes an unusual small C2-domain protein containing a Ca²⁺-binding module but lacking phospholipid-binding properties. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2011, 1810: 1317-1322.

[13] Nalefski E A, Falke J J. The C2 domain calcium-binding motif: Structure and functional diversity. Protein Science, 1996, 5: 2375-2390.

[14] Park N, Yoo J C, Lee Y S, Choi H Y, Hong S G, Hwang E M, Park J Y. Copine1 C2 domains have a critical calcium-independent role in the neuronal differentiation of hippocampal progenitor HiB5 cells. Biochemical and Biophysical Research Communications, 2014, 454: 228-233.

[15] Jean S, Zapata-Jenks M A, Farley J M, Tracye E, Mayer D G. Plasmodium falciparum double C2 domain protein, PfDOC2, binds to calcium when associated with membranes. Experimental Parasitology, 2014, 144: 91-95.

[16] Zhang D P, Aravind L. Identification of novel families and classification of the C2 domain superfamily elucidate the origin and evolution of membrane targeting activities in eukaryotes. Gene, 2010, 469: 18-30.

[17] Yang H, Li Y, Hua J. The C2 domain protein BAP1 negatively regulates defense responses in Arabidopsis. The Plant Journal, 2006, 48: 238-248

[18] Tiwari K, Paliyath G. Cloning, expression and functional characterization of the C2 domain from tomato phospholipase Dα. Plant Physiology and Biochemistry, 2011, 49: 18-32.

[19] Kim Y C, Kim S Y, Choi D, Ryu C M, Park J M. Molecular characterization of a pepper C2 domain-containing SRC2 protein implicated in resistance against host and non-host pathogens and abiotic stresses. Planta, 2008, 227: 1169-1179.

[20] Kim C Y, Koo Y D, Jin J B, Moon B C, Kang C H, Kim S T, Park B O, Lee S Y, Kim M L, Hwang I, Kang K Y, Bahk J D, Lee S Y, Cho M J. Rice C2-domain proteins are induced and translocated to the plasma membrane in response to a fungal elicitor. Biochemistry, 2003, 42: 11625-11633.

[21] Ouelhadj A, Kuschk P, Humbeck K. Heavy metal stress and leaf senescence induce the barley gene HvC2d1 encoding a calcium- dependent novel C2 domain-like protein. New Phytologist, 2006, 170: 261-273.

[22] Sakamoto M, Tomita R, Kobayashi K. A protein containing an XYPPX repeat and a C2 domain is associated with virally induced hypersensitive cell death in plants. FEBS Letters, 2009, 583: 2552-2556.

[23] 张兰军, 姬飞腾, 王丽丽, 亓岽东, 朱燕, 邓馨. 复苏植物旋蒴苣苔C2结构域小蛋白BhC2DP1参与植物对ABA的反应. 植物学报, 2012, 47: 11-27.

Zhang L J, Ji F T, Wang L L, Qi D D, Zhu Y, Deng X. A Small C2-domain protein from the resurrection plant Boea hygrometrica promotes plant responses to abscisic acid. Chinese Bulletin of Botany,2012, 47: 11-27. (in Chinese)

[24] Verwoerd T C, Dekker B M, Hoekema A. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research, 1989, 17: 2362.

[25] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods, 2001, 25: 402-408.

[26] Hetherington A M, Brownlee C. The generation of Ca²⁺ signals in plants. Annual Review of Plant Biology, 2004, 55: 401-427.

[27] Gerke V, Moss S E. Annexins: From structure to function. Physiological Reviews, 2002, 82: 331-371.

[28] Yokotani N, Ichikawa T, Kondou Y, Maeda S, Iwabuchi M, Mori M, Hirochika H, Matsui M, Oda K. Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to abiotic and biotic stresses in transgenic Arabidopsis. Plant Molecular Biology, 2009, 71: 391-402.

[29] Kim Y J, Kim J E, Lee J H, Lee M H, Jung H W, Bahk Y Y, Hwang B K, Hwang I, Kim W T. The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.). FEBS Letters, 2004, 556: 127-136.

[30] Bowler C, Fluhr R. The role of calcium and activated oxygens as signals for controlling cross-tolerance. Trends in Plant Science, 2000, 5: 241-246.

[31] Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K. Crosstalk between abiotic and biotic stress responses: A current view from the points of convergence in the stress signaling networks. Current Opinion in Plant Biology, 2006, 9: 436-442.

[32] Schapire A L, Voigt B, Jasik J, Rosado A, Lopez-Cobollo R, Menzel D, Salinas J, Mancuso S, Valpuesta V, Baluska F, Botella M A. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability. The Plant Cell , 2008, 20:3374-3388.