棉花钠尿肽基因GhPNP1的耐旱功能分析

doi:10.3864/j.issn.0578-1752.2015.12.003

摘要/Abstract

摘要： 【目的】分析棉花中钠尿肽GhPNP1的结构特征、表达模式以及耐旱功能，并分析其耐旱机制，为将该基因应用于作物改良奠定基础。【方法】通过对从植物中水平转移到大丽轮枝菌中的钠尿肽基因AVE1进行同源性搜索，得到与AVE1蛋白序列相似度较高的其他物种的蛋白序列；使用MEGA5软件对AVE1蛋白序列及其同源序列进行多序列比对分析并构建同源物种间系统进化树；利用MEGA和expasy在线工具进行蛋白序列分析；并根据编码该蛋白的核酸序列设计引物在陆地棉品种奥3503中克隆到其同源基因GhPNP1。使用多种生物信息学软件分析GhPNP1的分子特性，包括GhPNP1编码蛋白的等电点、分子量、信号肽、进化关系等进行预测分析。利用实时荧光定量PCR（qRT-PCR）分析GhPNP1在不同器官部位的组织表达模式以及受到PEG模拟干旱胁迫处理后的表达模式。将GhPNP1的cDNA序列连入CLCrV沉默载体中，构建GhPNP1的病毒诱导基因沉默载体CLCrV:GhPNP1，转入农杆菌，并通过和辅助载体CLCrVB共侵润2叶期幼苗进行叶片注射，获得GhPNP1的沉默植株。利用PEG模拟干旱处理沉默植株检测其耐旱性，并测定沉默植株的失水率、相对含水量、丙二醛（MDA）含量、总抗氧化活性（T-AOC水平）、离子渗漏率等与植物抗逆相关的生理指标。【结果】从陆地棉奥3503中克隆到的GhPNP1的开放阅读框长度为396 bp，编码131个氨基酸，信号肽长度为15个氨基酸，通过系统进化树分析GhPNP1编码的蛋白含有保守的钠尿肽结构域，与可可树的PNP蛋白进化关系最近。GhPNP1在棉花植株的根、茎、叶中均表达且在茎中表达量较高，PEG模拟干旱处理后根、茎、叶中的GhPNP1均上调表达。GhPNP1沉默后棉花植株耐旱性显著降低。在干旱条件下，GhPNP1沉默植株的MDA含量、离子渗漏率、叶片失水率均高于对照的未沉默植株；而沉默植株的总抗氧化能力（T-AOC水平）、相对含水量显著低于对照的未沉默植株。【结论】从棉花中克隆得到一个植物钠尿肽基因，受干旱胁迫诱导上调表达，沉默后耐旱性降低。推测GhPNP1可能通过cGMP信号途径参与棉花的干旱胁迫，在干旱胁迫下对棉花耐旱性起正调控作用。

关键词: 陆地棉, 病毒诱导的基因沉默, 干旱胁迫, 功能分析

Abstract: 【Objective】 The objectives of this research are to analyze the structural features, patterns of expression and drought tolerance functions of natriuretic peptide gene GhPNP1 in cotton, and provide a theoretical basis for future deeper study of crop drought tolerance. 【Method】 The homolog protein sequences of plant natriuretic peptides in cotton were obtained by a BLASTP search in Gossypium raimondii protein database using the query protein sequence of Verticillium dahliae plant natriuretic peptides gene Ave1 acquired from plants through horizontal gene transfer. Homologous analysis and multiple alignments were performed with MEGA 5. Expasy online tools were used for protein sequence analysis. The GhPNP1 was amplified in Gossypium hirsutum Ao3503 using the primers that were designed according to the nucleotide acid sequence of the homolog protein sequence. The isoelectric point, molecular weight, signal peptide and phylogenetic tree of the encoding protein were analyzed by related bioinformatics programs. The expression analysis of GhPNP1 gene in different organs and by PEG simulating drought stress treatment was conducted by real-time PCR. The virus induced gene silence vector was constructed and the GhPNP1 silenced cotton plants were obtained. The cDNA sequences of GhPNP1 was add to CLCrV silencing vector to construct virus-induced gene silencing vector CLCrV: GhPNP1 of GhPNP1. At 2-leaf-stage, cotton plant was infected and leaf tissues were obtained from hormone treated plant. PEG simulating drought stress treatment was used to determine the drought tolerance of GhPNP1 silenced cotton plants and test the several physiological indexes related to stress tolerance, such as water loss rate, relative water content, MDA content, T-AOC level and electrolyte leakage in GhPNP1 silenced cotton plants. 【Result】 GhPNP1 obtained in Gossypium hirsutum Ao3503 has an ORF with 396 nucleotides and encodes a protein of 131 amino acids, its isoelectric point is 9.13, the predicted molecular weight is 14.6 kD and with a signal peptide of 15 amino acids. The protein contained a conserved natriuretic peptides domain and with the highest similarity of GhPNP1 in Theobroma cacao. Quantitative real-time PCR (qRT-PCR) was used to examine the expression pattern of GhPNP1 in drought stress treatment. GhPNP1 exhibited weakly organ different expressed pattern with a little stronger expressed in stem, and moderately expressed in cotton root and leaves. In addition, PEG simulating drought stress treatment induced the up-regulated expression of GhPNP1 significantly in all the organs. The drought tolerance of GhPNP1 silenced cotton plants decreased significantly. The MDA content, electrolyte leakage and water loss rate of GhPNP1 silenced cotton plants were significantly higher than un-silenced cotton plants, wherever the T-AOC level and relative water content of silenced cotton plants were significantly lower than the un-silenced cotton plants. 【Conclusion】A plant natriuretic peptides gene (GhPNP1) was cloned from cotton, which is induced up-regulated by PEG simulating drought stress treatment and GhPNP1 silenced cotton plants significantly decreased drought tolerance. These imply that GhPNP1 may affect the drought tolerance of cotton through a cGMP dependent regulation pathway. GhPNP1 may play a positive role in drought tolerance of cotton.

Key words: upland cotton, virus induced gene silence, drought stress, function analysis

刘小双，刘廷利，袁洪波，张保龙，王荣富. 棉花钠尿肽基因GhPNP1的耐旱功能分析[J]. 中国农业科学, 2015, 48(12): 2306-2316.

LIU Xiao-shuang, LIU Ting-li, YUAN Hong-bo, ZHANG Bao-long, WANG Rong-fu. Drought-Enduring Functional Analysis of a Natriuretic Peptide Gene GhPNP1 in Cotton[J]. Scientia Agricultura Sinica, 2015, 48(12): 2306-2316.

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参考文献

[1] 李东晓, 李存东, 孙传范, 孙红春, 刘连涛, 张永江, 肖凯. 干旱对棉花主茎叶片内源激素含量与平衡的影响. 棉花学报, 2010, 22(3): 231-235.

Li D X, Li C D, Sun C F, Sun H C, Liu L T, Zhang Y J, Xiao K. The effects of drought on endogenous hormone contents and balance in main stem leaves of cotton. Cotton Science, 2010, 22(3): 231-235. (in Chinese)

[2] de Bold A J, Borenstein H B, Veress A T, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Science, 1981, 28(1): 89-94.

[3] Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature,1988, 332(6159): 78-81.

[4] Sudoh T, Minamino N, Kangawa K, Matsuo H. C-Type natriuretic peptide(CNP): A new member of natriuretic peptide family identified in porcine brain. Biochemical and Biophysical Research Communications, 1990, 168(2): 863-870.

[5] Feller S M, Gagelmann M, Forssmann W G. Urodilatin-a newly described member of the ANP family. Trends in Pharmacological Sciences, 1989, 10(3): 93-94.

[6] 张春荣, 李玲. 植物中的钠尿肽系统. 植物生理学通讯, 2001, 37(3): 250-254.

Zhang C R, Li L. Natriuretic peptides system in plants. Plant Physiology Communications, 2001, 37(3): 250-254. (in Chinese)

[7] Gehring C A, Irving H R. Natriuretic peptides-a class of heterologous molecules in plants. International Journal of Biochemistry and Cell Biology,2003, 35(9): 1318-1322.

[8] Morse M, Pironcheva G, Gehring C. AtPNP-A is a systemically mobile natriuretic peptide immunoanalogue with a role in Arabidopsis thaliana cell volume regulation. FEBS Letters, 2004, 556(1/3): 99-103.

[9] Gottig N, Garavaglia B S, Daurelio L D, Valentine A, Gehring C, Orellana E G, Ottado J. Xanthomonas axonopodis pv. citri uses a plant natriuretic peptide-like protein to modify host homeostasis. Proceedings of the National Academy of Sciences of the United States of America6., 2008, 105(47): 18631-1863

[10] Garavaglia B S, Thomas L, Zimaro T, Gottig N, Daurelio L D, Ndimba B, Orellano E G, Ottado J, Gehring C. A plant natriuretic peptide-like molecule of the pathogen Xanthomonas axonopodis pv. citri causes rapid changes in the proteome of its citrus host. BMC Plant Biology, 2010, DOI: 10.1186/1471-2229-10-51.

[11] Wang Y H, Gehring C, Cahill D M, Irving H R. Plant natriuretic peptide active site determination and effects on cGMP and cell volume regulation. Functional Plant Biology, 2007, 34(7): 645-653.

[12] Rafudeen S, Gxaba G, Makgoke G, Bradley G, Pironcheva G, Raitt L, Irving H, Gehring C. A role for plant natriuretic peptide immuno- analogues in NaCl and droughtstress responses. Physiologia Plantarum,2003, 119(4): 554-562.

[13] Wang Y H, Gehring C, Irving H R. Plant natriuretic peptides are apoplastic and paracrine stress response molecules. Plant Cell Physiology, 2011, 52(5): 837-850.

[14] de Jonge R, van Esse H P, Maruthachalam K, Bolton M D, Santhanam P, Saber M K, Zhang Z, Usami T, Lievens B, Subbarao K V. Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(13): 5110-5115.

[15] 杨召恩, 杨作仁, 刘坤, 刘传亮, 张朝军, 李付广. 一个亚洲棉MYB家族新基因的克隆及特征分析. 中国农业科学, 2013, 46(1): 195-204.

Yang Z E, Yang Z R, Liu K, Liu C L, Zhang C J, Li F G. Cloning and characterization of a novel gene of MYB family from Gossypium arboreum L.. Scientia Agricultura Sinica, 2013, 46(1): 195-204. (in Chinese)

[16] 李智元, 刘锦春. 植物响应干旱的生理机制研究进展. 西藏农业科技, 2010(3): 12-19.

Li Z Y, Liu J C. Prospects of mechanism of drought tolerance in plant. Journal of Agricultural Sciences, 2010(3): 12-19. (in Chinese)

[17] Hall B G. Building phylogenetic trees from molecular data with MEGA. Molecular Biology and Evolution, 2013, 30(5): 1229-1235.

[18] 房栋, 吕俊宏, 郭旺珍, 张天真. 一个新的棉花MYB类基因（GhTF1）的克隆及染色体定位分析. 作物学报, 2008(2): 207-211.

Fang D, Lü J H, Guo W Z, Zhang T Z. Cloning and mapping of a new MYB transcription factor (GhTF1) in cotton. The Crop Journal, 2008(2): 207-211. (in Chinese)

[19] 张雪妍, 刘传亮, 王俊娟, 李付广, 叶武威. PEG胁迫方法评价棉花幼苗耐旱性研究. 棉花学报, 2007(3): 205-209.

Zhang X Y, Liu C L, Wang J J, Li F G, Ye W W. Evaluation to the drought tolerance of cotton by PEG water-stress. Cotton Science, 2007(3): 205-209. (in Chinese)

[20] Anandalakshmi R, Pruss G J, Ge X, Marathe R, Mallory A C, Smith T H, Vance V B. A viral suppressor of gene silencing in plants. Proceedings of the National Academy of Sciences of the United States of America , 1998, 95(22): 13079-13084.

[21] Zhang B L, Yang Y W, Chen T Z, Yu W G, Liu T L, Hong J. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae. PloS One, 7(12): e51091.

[22] Zhou S, Hu W, Deng X, Ma Z, Chen L, Huang C, Wang C, Wang J, He Y, Yang G. Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco. PloS One, 2012, 7(12): e52439.

[23] 高俊凤. 植物生理学实验指导. 北京: 高等教育出版社, 2006.

Gao J F. Experimental Guidance for Plant Physiology. Beijing: Higher Education Press, 2006. (in Chinese)

[24] Yue Y, Zhang M, Zhang J, Tian X, Duan L, Li Z. Overexpression of the AtLOS5 gene increased abscisic acid level and drought tolerance in transgenic cotton. Journal of Experimental Botany, 2012, 63(10): 3741-3748.

[25] Hasegawa P M, Bressan R A, Zhu J K, Bohnert H J. Plant cellular and molecular responses to high salinity. Annual Reviews Plant Physiology Plant Molecule Biology, 2000, 51(1): 463-499.

[26] Yu X, Tang J, Wang Q, Ye W, Tao K, Duan S, Lu C, Yang X, Dong S. The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death. New Phytologist, 2012, 196(1): 247-260.

[27] Wei M, Wu Y, Chen D, Gu Y. Changes of free radicals and digestive enzymes in saliva in cases with deficiency in spleen-yin syndrome. Journal of Biomedical Research, 2010, 24(3): 250-255.

[28] Vesely D L, Gower W R, Giordano A T. Atrial natriuretic peptides are present throughout the plant kingdom and enhance solute flow in plants. The American Journal of Physiology,1993, 265(3): 465-477.

[29] Gu Z, Huang C, Li F, Zhou X. A versatile system for functional analysis of genes and micro-RNAs in cotton. Plant Biotechnology Journal,2014, 12(05): 638-649.

[30] Gaxiola R A, Li J, Undurraga S, Dang L M, Allen G J, Alper S L, Fink G R. Drought- and salt-tolerant plants result from overexpression of the AVP1 H⁺-pump. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(20): 11444-11449.