霜霉威胁迫下黄瓜CsWRKY30表达及功能分析

doi:10.3864/j.issn.0578-1752.2016.07.006

摘要/Abstract

摘要： 【目的】从前期转录组测序结果中筛选获得一个在霜霉威(propamocarb)胁迫条件下差异上调表达的基因CsWRKY30，对其进行克隆并分析其在霜霉威胁迫下的功能,了解黄瓜低霜霉威残留的分子机制。【方法】通过PCR技术扩增CsWRKY30全长，利用NCBI和PlantCARE在线工具分别进行该基因编码蛋白的保守结构域分析和启动子序列分析；利用实时荧光定量PCR分析CsWRKY30在霜霉威胁迫及其他胁迫条件下的相关表达模式；通过与GFP蛋白融合对CsWRKY30蛋白进行亚细胞定位；通过花序侵染法将CsWRKY30构建的植物过表达载体转化到哥伦比亚野生型拟南芥中，对获得的纯合转基因株系在霜霉威胁迫条件下的功能进行鉴定。【结果】CsWRKY30的CDS序列全长为1 014 bp，其编码的337个氨基酸中包含1个由60个氨基酸组成的WRKY结构域。CsWRKY30表达模式分析结果显示，黄瓜遭受霜霉威胁迫时，CsWRKY30在低霜霉威残留品系D0351中表达量显著上调，而在高霜霉威残留品系D9320中表达量并没有发生改变；在霜霉威胁迫的0.5—9 h间，该基因在D0351中的表达量一直明显高于对照，然而在24 h以后，该基因的表达不再显著上调。组织特异性表达分析表明，CsWRKY30主要在黄瓜果实中表达。蛋白亚细胞定位结果表明，CsWRKY30定位于细胞核。对CsWRKY30转基因拟南芥进行霜霉威胁迫发现，在未处理条件下，CsWRKY30 转基因拟南芥与野生型拟南芥表型上无明显差异；在2 mmol?L^-1霜霉威处理条件下，CsWRKY30转基因拟南芥萌发率及主根长均明显高于野生型拟南芥。在其他逆境作用下，CsWRKY30对霜霉威和多主棒孢霉菌条件积极响应，对干旱和高盐没有作用，同时受到脱落酸（ABA）信号诱导。【结论】黄瓜CsWRKY30在霜霉威胁迫条件下发挥重要作用，过量表达CsWRKY30可显著提高转基因拟南芥对霜霉威胁迫的抵抗能力。

关键词: 黄瓜, CsWRKY30, 霜霉威, 功能分析

Abstract: 【Objective】The objective of this study is to screen, clone and analyze the function of the cucumber transcription factor gene CsWRKY30, which was up-regulated under propamocarb stress in previous transcriptome analyses, CsWRKY30, and to understand the molecular mechanism of low propamocarb residue in cucumbers.【Method】The full length of CsWRKY30 was cloned by PCR, and protein sequences and promoter region of the gene were analyzed with NCBI and PlantCARE separately. The real-time PCR was used to analyze the expression pattern of CsWRKY30 under different treatments. The subcellular localization of CsWRKY30 was conducted through fusion with GFP protein. CsWRKY30 was over-expressed in Arabidopsis by the inflorescence infection method and got pure transgenic lines to identify gene function.【Result】The full length of CsWRKY30 was 1 014 bp and encoded 337 amino acids, which contained a WRKY conservative domain consisting of 60 amino acids. Expression pattern analysis showed that CsWRKY30 was induced by propamocarb stress in the low propamocarb residue D0351, while there was no differences in the high propamocarb residue D9320. The expression level of CsWRKY30 in D0351 was significantly higher than that in the control during 0.5-9 h, while the expression of this gene was no longer significantly up-regulated after 24 h. Tissue specific expression analysis showed that CsWRKY30 was mainly expressed in cucumber fruits. Subcellular localization indicated that CsWRKY30 was mainly located on the nucleus in plant cells. Over-expression CsWRKY30 Arabidopsis under propamocarb stress test showed that there were no significant phenotypic differences between transgenic Arabidopsis and wild types under the untreated condition, while under 2 mmol?L^-1 propamocarb stress the transgenic Arabidopsis germination rate and root length were apparently higher than that of the wild type. Expression of CsWRKY30 also showed a trend of increasing under Corynespora cassiicola treatment and was induced by abscisic acid (ABA), while showing no effects in drought and high salt stresses.【Conclusion】CsWRKY30 played an important role under propamocarb stress in cucumber, and over-expression of CsWRKY30 enhanced transgenic plants tolerance.

Key words: cucumber, CsWRKY30, propamocarb, function analysis

李胜男，秦智伟，辛明，周秀艳. 霜霉威胁迫下黄瓜CsWRKY30表达及功能分析[J]. 中国农业科学, 2016, 49(7): 1277-1288.

LI Sheng-nan, QIN Zhi-wei, XIN Ming, ZHOU Xiu-yan. Expression and Functional Analysis of CsWRKY30 in Cucumber Under Propamocarb Stress[J]. Scientia Agricultura Sinica, 2016, 49(7): 1277-1288.

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.0578-1752.2016.07.006

https://www.chinaagrisci.com/CN/Y2016/V49/I7/1277

参考文献

[1] 张鑫, 张哲, 范春朴. 日光温室黄瓜霜霉病的发生原因及防治对策. 现代农业科技, 2013(4): 149.

Zhang X, Zhang Z, Fan C P. The occurrence cause and control countermeasure of cucumber downy mildew in sunlight greenhouse. Modern Agricultural Sciences and Technology, 2013(4): 149. (in Chinese)

[2] 禹化强. 大棚黄瓜主要病害的防治研究. 中国果菜, 2015, 35(2): 48-52.

Yu H Q. Research on the prevention and control of major diseases of cucumber in greenhouse. China Fruit and Vegetable, 2015, 35(2): 48-52. (in Chinese)

[3] 柳亚男, 李敏敏, 范蓓, 卢嘉, 贺妍, 孔志强, 孙玉凤, 王凤忠. 超高效液相色谱串联质谱法测定霜霉威在番茄和土壤中残留及消解动态. 环境化学, 2015, 34(6): 1072-1077.

Liu Y N , Li M M, Fan B, Lu J, He Y, Kong Z Q, Sun Y F, Wang F Z. Residues and dissipation of propamocarb in tomatoes and soil using UPLC-MS/MS. Environmental Chemistry, 2015, 34(6): 1072-1077. (in Chinese)

[4] Zhang Z Y, Liu X J, Hong X Y. Effects of home preparation on pesticide residues in cabbage. Food Control, 2007, 18: 1484-1487.

[5] 万阳芳, 李慧颖, 刘俊果, 郝建雄. 农产品中农药残留化学降解方法研究进展. 河北工业科技, 2014, 31(2): 148-151.

Wan Y F, Li H Y, Liu J G, Hao J X. Review on the chemical degradation methods of pesticide residues in agricultural products. Hebei Journal of Industrial Science and Technology, 2014, 31(2): 148-151. (in Chinese)

[6] Xia X J, Zhang Y, Wu J X, Wang J T, Zhou Y H, Shi K, Yu Y L, Yu J Q. Brassinosteroids promote metabolism of pesticides in cucumber. Journal of Agricultural and Food Chemistry, 2009, 57(18): 8406-8413.

[7] 王梅, 段劲生, 孙明娜, 张勇, 胡本进, 高同春. 农产品中农药残留去除技术研究进展. 农药, 2007, 46(7): 442-446.

Wang M, Duan J S, Sun M N, Zhang Y, Hu B J, Gao T C. Research progress on techniques of reducing pesticide residues in agro-product. Agrochemicals, 2007, 46(7): 442-446. (in Chinese)

[8] 刘芳芳. 黄瓜种质资源低农药残留性评价[D]. 哈尔滨: 东北农业大学, 2008.

Liu F F. Study on evaluation of cucumber germplasm resources with low pesticide residue content[D]. Harbin: Northeast Agricultural University, 2008. (in Chinese)

[9] 马佰慧. 黄瓜果实霜霉威残留性的遗传分析与SSR分子标记[D]. 哈尔滨: 东北农业大学, 2010.

Ma B H. Genetic analysis and molecular markers for propamocarb residues in cucumber[D]. Harbin: Northeast Agricultural University, 2010. (in Chinese)

[10] Wu P, Qin Z W, Zhao W, Zhou X Y, Wu T, Xin M, Guo Q Q. Transcriptome analysis reveals differentially expressed genes associated with propamocarb response in cucumber (Cucumis sativus L.) fruit. Acta Physiologiae Plantarum, 2013, 35(8): 2393-2406.

[11] Xu X H, Zhao J H, Liu Q L, Frank T, Engel K H, An G, Shu Q Y. Mutations of the multi-drug resistance-associated protein ABC transporter gene 5 result in reduction of phytic acid in rice seeds. Theoretical and Applied Genetics, 2009, 119(1): 75-83.

[12] Giraudo M, Hilliou F, Fricaux T, Audant P, Feyereisen R, Le Goff G. Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides. Insect Molecular Biology, 2015, 24(1): 115-128.

[13] Eulgem T, Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors. Trends in Plant Science, 2000, 5(5): 199-206.

[14] Peng X X, Hu Y J, Tang X K, Zhou P L, Deng X B, Wang H H, Guo Z J. Constitutive expression of rice WRKY30 gene increases the endogenous jasmonic acid accumulation, PR gene expression and resistance to fungal pathogens in rice. Planta, 2012, 236(5): 1485-1498.

[15] Scarpeci T E, Zanor M I, Mueller-Roeber B, Valle E M. Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana. Plant Molecular Biology, 2013, 83(3): 265-277.

[16] Sun X C, Gao Y F, Li H R, Yang S Z, Liu Y S. Over-expression of SlWRKY39 leads to enhanced resistance to multiple stress factors in tomato. Journal of Plant Biology, 2015, 58(1): 52-60.

[17] Dang F F, Wang Y N , Yu L, Eulgem T, Lai Y, Liu Z Q, Wang X, Qiu A L, Zhang X T, Lin J, Chen Y S, Guan D Y, Cai Y H, Mou L S, He L S. CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant Cell and Environment, 2013, 36(4): 757-774.

[18] Jiang Y J, Liang G, Yang S, Yu D. Arabidopsis WRKY57 functions as a node of convergence for jasmonic acid- and auxin-mediated signaling in jasmonic acid-induced leaf senescence. The Plant Cell, 2014, 26(1): 230-245.

[19] Chen H, Lai Z B, Shi J W, Xiao Y, Chen Z X, Xu X P. Roles of arabidopsisWRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biology, 2010, 10(1): 281.

[20] Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols, 2007, 2(7): 1565-1572.

[21] Bechtold N, Pelletier G. In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Metholds in Molecular Biology, 1998, 82: 259-266.

[22] Wang C, Deng P Y, Chen L L, Wang X T, Ma H, Hu W, Yao N G, Feng Y, Chai R H, Yang G X, He G Y. A wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco. Plos One, 2013, 8(6): e65120.

[23] Zheng Z Y, Qamar S A, Chen Z X, Mengiste T. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. The Plant Journal, 2006, 48(4): 592-605.

[24] 吴鹏, 郭茜茜, 武涛, 秦智伟. 黄瓜ABC转运蛋白(abca19)的克隆及其对农药霜霉威的胁迫响应. 作物杂志, 2015(3): 45-51.

Wu P, Guo Q Q, Wu T, Qin Z W. Cloning of ABC transporter protein (abca19) in Cucumis sativus L. and response under propamocarb stress. Crops, 2015(3): 45-51. (in Chinese)

[25] Li J B, Luan Y S. Molecular cloning and characterization of a pathogen-induced WRKY transcription factor gene from late blight resistant tomato varieties Solanum pimpinellifolium L3708. Physiological and Molecular Plant Pathology, 2014, 87: 25-31.

[26] Wang Y N, Dang F F, Liu Z Q, Wang X, Eulgem T, Lai Y, Yu L, She J J, You L, Lin J H, Chen C C, Guan D Y, Qiu A L, He S L. CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection. Molecular Plant Pathology, 2013, 14(2): 131-144.

[27] Xu Q, Feng W J, Peng H R, Ni Z P, Sun Q X. TaWRKY71, a WRKY transcription factor from wheat, enhances tolerance to abiotic stress in transgenic Arabidopsis thaliana. Cereal Research Communications, 2014, 42(1): 47-57.

[28] Luo X, Bai X, Sun X S, Zhu D, Liu B H, Ji W, Cao L, Wu J, Hu M, Liu X, Tang L L, Zhu Y M. Expression of wild soybean WRKY20 in Arabidopsis enhances drought tolerance and regulates ABA signalling. Journal of Experimental Botany, 2013, 64(8): 2155-2169.

[29] Shang Y, Yan L, Liu Z Q, Cao Z, Mei C, Xin Q, Wu F Q, Wang X F, Du S Y, Jiang T, Zhang X F, Zhao R, Sun H L, Liu R, Yu Y T, Zhang D P. The Mg-chelatase H subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition. The Plant Cell, 2010, 22(6): 1909-1935.

[30] 吴鹏. 黄瓜低霜霉威残留性的生理生化及分子基础研究[D]. 哈尔滨: 东北农业大学, 2013.

Wu P. Studies on physiological and biochemical and molecular basis of low propamocarb residue in cucumber[D]. Harbin: Northeast Agricultural University, 2013. (in Chinese)

[31] Ling J, Zhang Y, Yu H J, Mao Z C, Gu X F , Huang S W, Xie B Y. Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genomics, 2011, 12: 471.