小麦逆境胁迫相关基因Ta14S的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2012.06.022

摘要/Abstract

摘要： 【目的】克隆与逆境胁迫相关的基因，通过对目的基因的表达分析进一步解析植物的抗逆机制，为小麦抗逆育种提供候选基因和理论依据。【方法】基于cDNA芯片数据获得的水分胁迫诱导上调表达基因EST序列，运用RACE技术进行cDNA全长克隆，采用生物信息学软件分析克隆基因的编码蛋白特性，并利用实时荧光定量PCR分析该基因在不同组织及不同胁迫处理条件下的表达模式。【结果】通过RACE扩增获得小麦cDNA全长序列（GenBank登录号：JN650603），命名为Ta14S。该基因序列全长为1 056 bp，其中，5′端非编码区11 bp，3′端非编码区253 bp，开放阅读框为792 bp，编码263个氨基酸。序列比对发现其蛋白质序列包含1个蛋白激酶C的底物结构域、1个类膜蛋白结合域、1个转录因子结合域和1个核输出信号结合域，具有植物14-3-3蛋白的结构特征；运用实时荧光定量PCR进行Ta14S表达分析，该基因在小麦苗期根中表达量最高，在PEG和低温胁迫的任何时间点均稳定上调表达，在ABA和高温胁迫的6 h内其相对表达量均显著高于对照，推测Ta14S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Ta14S的全长cDNA序列，其编码蛋白包含与蛋白质互作的典型功能域；通过对Ta14S在干旱、高温、低温、ABA胁迫过程中的表达特性分析表明，Ta14S在小麦逆境胁迫中发挥着重要的调控功能。

关键词: 小麦, 14-3-3蛋白, 逆境胁迫, 表达

Abstract: 【Objective】In this study, the objective is to clone the stress resistance-related genes, and analyze the expression pattern of those genes which aim to investigate the underlying molecular mechanism of stress resistance and provide candidate genes for stress-resistance plant breeding.【Method】 Based on the analysis of up-regulated EST obtained by cDNA chip, a full-length cDNA sequence was cloned from wheat through rapid amplification of cDNA ends (RACE) method and proper bioinformatics softwares were applied for characterizing the cloned genes and the deduced proteins. The gene expression characters were analyzed by the real time PCR.【Result】The full-length cDNA sequence designated as Ta14S from wheat was 1 056 bp in length, contains a 792 bp open reading frame (ORF) encoding a 263 amino acid proteins, with 11 bp in the 5' UTR and 253 bp in the 3' UTR. Similarity alignment of plant 14-3-3 proteins by DNAMAN revealed four high conserved domains. These domains were a potential substrate for protein kinase C, annexin-like domain, domain to bind transcription factor and nuclear export signal peptide, respectively. This result suggests that the Ta14S belongs to the family of 14-3-3. Real time PCR analysis revealed that the expression level of Ta14S was the highest in root than in leave and stem. The expression of Ta14S was steadily up-regulated in any of the time points under PEG and low temperature stress and the relative expression level was significantly higher than those of control within 6 h of ABA and low temperature stress treatments. This result suggests that Ta14S may be involved in stress resistance-related response of ABA signaling pathways in wheat roots.【Conclusion】A full-length cDNA of Ta14S was cloned from wheat and the typical protein interact binding domains were found in the deduced proteins. The expression patterns of Ta14S gene under drought, high and low temperature and ABA treatments showed that Ta14S might play an important regulation role under stress in wheat. The results provided important information for further studies on molecular regulation mechanism underlying abiotic stress resistance in wheat.

Key words: wheat (Triticum aestivum), 14-3-3 protein, stress, expression

任江萍, 刘海伦, 王新国, 牛洪斌, 李永春, 王翔, 陈新, 尹钧. 小麦逆境胁迫相关基因Ta14S的克隆及表达分析[J]. 中国农业科学, 2012, 45(6): 1226-1234.

REN Jiang-Ping, LIU Hai-Lun, WANG Xin-Guo, NIU Hong-Bin, LI Yong-Chun, WANG Xiang, CHEN Xin, YIN Jun. Cloning and Expression Analysis of A Stress-related Ta14S Gene from Wheat[J]. Scientia Agricultura Sinica, 2012, 45(6): 1226-1234.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.06.022

https://www.chinaagrisci.com/CN/Y2012/V45/I6/1226

参考文献

[1]刘录祥, 赵林姝, 梁欣欣, 郑企成, 刘强, 王晶, 郭会君, 赵世荣, 陈文华. 基因枪法获得逆境诱导转录因子DREB1A转基因小麦的研究. 中国生物工程杂志, 2003, 23(11): 53-56.

Liu L X, Zhao L S, Liang X X, Zheng Q C, Liu Q, Wang J, Guo H J, Zhao S R, Chen W H. Study on production of transgenic wheat with a stress-inducible transcription factor gene DREB1A by microprojectile bombardment. China Biotechnology, 2003, 23(11): 53-56. (in Chinese)

[2]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.

[3]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.

[4]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.

[5]Andleeb S, Amin I, Bashir A, Briddon R W, Mansoor S. Transient expression of betaC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions. Virology Journal, 2010, 7: 373.

[6]Chen W, Yao X Q, Cai K Z, Chen J N. 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.

[7]Ferl R J. 14-3-3 proteins and signal transduction. Annual Review of Plant Physiology and Plant Molecular Biology, 1996, 47: 49-73.

[8]Fu H, Subramanian R R, Masters S C. 14-3-3 Proteins: Structure function and regulation. Annual Review of Pharmacology and Toxicology, 2000, 40: 617- 647.

[9]Pertl H, Rittmann S, Schulze W X, Obermeyer G. Identification of lily pollen 14-3-3 isoforms and their subcellular and time-dependent expression profile. Biological Chemistry, 2011, 392: 249-262.

[10]Comparot S, Lingiah G, Martin T. Function and specificity of 14-3-3 proteins in the regulation of carbohydrate and nitrogen metabolism. Journal of Experimental Botany, 2003, 54: 595-604.

[11]Oh C S, Pedley K F, Martin G B. Tomato 14-3-3 protein 7 positively regulates immunity-associated programmed cell death by enhancing protein abundance and signaling ability of MAPKKKα. The Plant Cell, 2010, 22: 260-272.

[12]Fulgosi H, Soll J, Maraschin S de F, Korthout H A A J, Wang M, Testerink C. 14-3-3 proteins and plant development. Plant Molecular Biology, 2002, 50: 1019-1029.

[13]Roberts M R, Salinas J, Collinge D B. 14-3-3 proteins and the response to abiotic and biotic stress. Plant Molecular Biology, 2002, 50: 1031-1039.

[14]Bridges D, Moorhead G B G. 14-3-3 proteins: A number of functions for a numbered protein. Science’s STKE, 2004, 242: 1-8.

[15]Finni C, Andersen C H, Borch J, Gjetting S, Christensen A B, De Boer A H, Thordal-Christensen H, Collinge D B. Do 14-3-3 proteins and plasma membrane H+-ATPases interact in the barley epidermis in response to the barley powdery mildew fungus? Plant Molecular Biology, 2002, 49: 137-147.

[16]Mackintosh C. Regulation of plant nitrate assimilation from ecophysiology to brain proteins. New Phytologists, 1998, 139: 153-159.

[17]Moorhead G, Douglas P, Corelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, MacKintosh C. Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. The Plant Journal, 1999, 18: 1-12.

[18]Pan S, Sehnke P C, Ferl R J, Gurley W B. Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription whea part of a DNA binding complex. The Plant Cell, 1999, 1: 1591-1602.

[19]Jarillo J A, Capel J, Leyva A, Martínez-Zapater J M, Salinas J. Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators. Plant Molecular Biology, 1994, 25: 693-704.

[20]Chen F, Li Q, Sun L, He Z. The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress. DNA Research, 2006, 13: 53-63.

[21]倪中福, 孙其信, 吴利民. 普通小麦不同优势杂交种及其亲本之间基因表达差异比较研究. 中国农业大学学报, 2000, 5(1): 1-8.

Ni Z F, Sun Q X, Wu L M. Differential gene expression between wheat hybrids and their parental inbreds in seedling leaves of early and vigorous tillering stages. Journal of China Agricultural University, 2000, 5(1): 1-8. (in Chinese)

[22]Rosenquist M, Sehnke P, Ferl R J, Sommarin M, Larsson C. Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity? Journal of Molecular Evolution, 2000, 51: 446-458.

[23]Sehnke P C, Rosenquist M, Alsterfjord M, DeLille J, Sommarin M, Larsson C, Ferl R J. Evolution and isoform specificity of plant 14-3-3 proteins. Plant Molecular Biology, 2002, 50: 1011-1018.

[24]Yao Y, Du Y, Jiang L, Liu J Y. Molecular analysis and expression patterns of the 14-3-3 gene family from Oryza sativa. Journal of Biochemistry and Molecular Biology, 2007, 40: 349-357.

[25]Ikeda Y, Koizume N, Kusano T, Sano H. Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase. Journal of Biology Chemistry, 2000, 275: 31695-31700.

[26]Wang C, Ma Q H, Lin Z B, He P, Liu J Y. Cloning and characterization of a cDNA encoding 14-3-3 protein with leaf and stem-specific expression from wheat. DNA Sequence, 2008, 19: 130-136.

[27]Wijngaard P W, Sinnige M P, Roobeek I, Reumer A, Schoonheim P J, Mol J N M, Wang M, De Boer A H. Abscisic acid and 14-3-3 proteins control K+ channel activity in barley embryonic root. The Plant Journal, 2005, 41: 43-55.

[28]Shanko A V, Mesenko M M, Klychnikov O I, Nosov A V, Ivanov V B. Proton pumping in growing part of maize root: Its correlation with 14-3-3 protein content and changes in response to osmotic stress. Biochemistry Biokhimiia, 2003, 68: l320-1326.

[29]Chelysheva V V, Smolenskaya I N, Trofimova M C, Babakov A V, Muromtsev G S. Role of the 14-3-3 proteins in the regulation of H+-ATPase activity in the plasma membrane of suspension-cultured sugar beet cells under cold stress. FEBS Letters, 1999, 456: 22-26.

[30]Yan J, He C, Wang J, Mao Z, Holaday S A, Allen R D, Zhang H. Overexpression of the Arabidopsis 14-3-3 protein GF14 lambda in cotton leads to a "stay-green" phenotype and improves stress tolerance under moderate drought conditions. Plant and Cell Physiology, 2004, 45: 1007-1014.