马铃薯晚疫病菌应答WRKY基因的结构功能预测与植物表达载体构建
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摘要
WRKY是一类广泛参与高等植物各种抗逆调控活动的转录因子,可以通过激活下游相关信号转导途径调节自身的应激反应,进而增强植物抗逆性。本研究通过RNA-Seq从马铃薯(Solanum tuberosum)中筛选出一个晚疫病菌诱导WRKY转录因子基因StWRKY。采用RT-PCR技术获得该基因CDS全长,并对其进行序列分析及结构功能预测。根据StWRKY蛋白序列进行同源性搜索,得到与其蛋白序列相似度较高的其他物种的蛋白序列,使用MEGA7软件对St WRKY蛋白序列及其同源序列进行多序列比对分析并构建了系统进化树。利用在线工具ProtParam对StWRKY进行氨基酸理化性质分析;利用SMART在线工具进行蛋白序列分析;利用SWISS-MODEL在线工具和PredictProtein在线平台分别对StWRKY蛋白二级结构和三级结构进行分析。结果表明,StWRKY核苷酸序列CDS全长为957 bp,编码含318个氨基酸的蛋白质,预测蛋白分子量为36.17 k D,等电点为6.49。既不是分泌蛋白也不是膜蛋白,未发现跨膜区、信号肽和复合螺旋区。StWRKY三维空间结构主要由无规卷曲以及β-折叠组成。通过XcmⅠ酶切、连接将StWRKY装载到pCXSN载体上,构建了该基因的超表达载体pCXSN-StWRKY。StWRKY基因的克隆,为进一步从分子水平上验证其抗晚疫病的生物学功能以及揭示马铃薯生物胁迫抗逆机制奠定基础,并为马铃薯抗病育种提供新的基因资源。
WRKY is a kind of transcriptional factor that widely participates in the regulation of various stress resistant activities of higher plants, which can regulate its own stress response by activating downstream related signal transduction pathway to enhance plant stress resistance. In this study, a WRKY transcription factor gene StWRKY induced by Phytophthora infestans was screened from Solanum tuberosum by RNA-seq. The full-length CDS of the gene was obtained by RT-PCR, and the sequence analysis and structural function prediction were carried out. Homology search was performed according to StWRKY protein sequence, and protein sequences of other species with high similarity to its protein sequences were obtained. A multiple sequence alignment was analyzed and phylogenetic tree was constructed on StWRKY protein sequence and its homologous sequences by MEGA7. The physical and chemical properties of St WRKY were analyzed by online tool ProtParam. StWRKY protein sequence was analyzed by SMART online tools. The secondary structure and tertiary structure of StWRKY protein were analyzed by SWISS-MODEL online tool and PreidictProtein online platform, respectively. The results showed that the full-length CDS of StWRKY nucleotide sequence was 957 bp, encoding 318 amino acids,with the predicted molecular mass of 36.17 kD and the isoelectric point of 6.49. Neither a secreted protein nor a membrane protein, and no transmembrane region, signal peptide and complex helix region were found. The StWRKY three-dimensional space structure was mainly composed of random coils and β-sheets. The overexpression vector pCXSN-StWRKY was constructed by loading St WRKY on plasmid pCXSN through XcmⅠenzyme digestion and linkage. The cloning of StWRKY gene could provide a basis for further identification of the biological function of anti-late blight on the molecular level, to reveal the resistance mechanism of potato biotic stress and offer new genetic resources for the resistance breeding of potato.
WRKY is a kind of transcriptional factor that widely participates in the regulation of various stress resistant activities of higher plants, which can regulate its own stress response by activating downstream related signal transduction pathway to enhance plant stress resistance. In this study, a WRKY transcription factor gene StWRKY induced by Phytophthora infestans was screened from Solanum tuberosum by RNA-seq. The full-length CDS of the gene was obtained by RT-PCR, and the sequence analysis and structural function prediction were carried out. Homology search was performed according to StWRKY protein sequence, and protein sequences of other species with high similarity to its protein sequences were obtained. A multiple sequence alignment was analyzed and phylogenetic tree was constructed on StWRKY protein sequence and its homologous sequences by MEGA7. The physical and chemical properties of St WRKY were analyzed by online tool ProtParam. StWRKY protein sequence was analyzed by SMART online tools. The secondary structure and tertiary structure of StWRKY protein were analyzed by SWISS-MODEL online tool and PreidictProtein online platform, respectively. The results showed that the full-length CDS of StWRKY nucleotide sequence was 957 bp, encoding 318 amino acids,with the predicted molecular mass of 36.17 kD and the isoelectric point of 6.49. Neither a secreted protein nor a membrane protein, and no transmembrane region, signal peptide and complex helix region were found. The StWRKY three-dimensional space structure was mainly composed of random coils and β-sheets. The overexpression vector pCXSN-StWRKY was constructed by loading St WRKY on plasmid pCXSN through XcmⅠenzyme digestion and linkage. The cloning of StWRKY gene could provide a basis for further identification of the biological function of anti-late blight on the molecular level, to reveal the resistance mechanism of potato biotic stress and offer new genetic resources for the resistance breeding of potato.
引文
Che n L.,Song Y.,Li S.,Zhang L.P.,Zou C.S.,and Yu D.Q.,2012,The role of WRKY transcription factors in plant abiotic stresses,Biochim.Biophys.Acta,1819(2):120-128
Eulgem T.,Rushton P.J.,Robatzek S.,and Somssich I.E.,2000,The WRKY superfamily of plant transcription factors,Trends Plant Sci.,5(5):199-206
Huang S.X.,and Liu Y.S.,2013,Genome-wide analysis of WRKY transcription factors in solanum tuberosum,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Ap plied&Environmental Biology),19(2):205-214(黄胜雄,刘永胜,2013,土豆WRKY转录因子家族的生物信息学分析,应用与环境生物学报,19(2):205-214)
Joshi R.,Wani S.H.,Singh B.,Boohra A.,Dar Z.A.,Lone A.A.,Pareek A.,and Singla-Pareek S.L.,2016,Transcription factors and plants response to drought stress:current understanding and future directions,Front Plant Sci.,7:1029
Li R.,and Lou Y.G.,2011,Research advances on stress responsive WRKY transcription factors in plants,Shengtai Xuebao(Acta Ecologica Sinica),31(11):3223-3231(李冉,娄永根,2011,植物中逆境反应相关的WRKY转录因子研究进展,生态学报,31(11):3223-3231)
Li X.,Chen Y.F.,and Yang X.,2017,Research progress of WRKY transcription factors in solanaceae plants,Yuanyi Xuebao(Acta Horticulturae Sinica),44(1):170-178(李笑,成玉富,杨旭,2017,茄科植物WRKY转录因子的研究进展,园艺学报,44(1):170-178)
Maeo K.,Hayashi S.,Kojima-Suzuki H.,Kojima-Suzuki H.,Morikami A.,and Nakamura K.,2001,Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins,Biosci.Biotechnol.Biochem.,65(11):2428-2436
Ren Y.J.,Wang H.X.,Li Y.J.,Chen Y.L.,Wang J.,and Tian Z.D.,2015,An earlyβ-aminobutyric acid responsive gene St WRKY5 confers resistance to late blight in potato,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(6):1207-1213(任亚娟,王海霞,李亚军,陈艳林,王静,田振东,2015,β-氨基丁酸早期诱导表达基因St WRKY5参与马铃薯晚疫病抗性,分子植物育种,13(6):1207-1213)
Wang T.,Ma S.,Sun J.Y.,and Ru J.R.,2017,Research progress in physiological race of Phytophthora infestans in China,Zhongguo Malingshu(Chinese Potato Journal),31(1):45-53(王腾,马爽,孙继英,汝甲荣,2017,中国马铃薯晚疫病菌生理小种研究进展,中国马铃薯,31(1):45-53)
Wen F.,Wu X.Z.,Liao L.,Liu X.S.,and Li P.,2017,Research advances on physiological function of WRKY transcription factor in plant stress resistance,Guangxi Zhiwu(Guihaia),7(1):69-79(文锋,吴小祝,廖亮,刘新圣,李鹏,2017,WRKY转录因子在植物抗逆生理中的研究进展,广西植物,7(1):69-79)
Xie Z.,Zhang Z.L.,Zou X.L.,Huang J.,Ruas P.,Thompson D.,and Shen Q.J.,2005,Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells,Plant Physiol.,137(1):176-189
Yang Z.H.,Yu Q.,and Liu H.G.,2017,Situation of potato industry in recent years and its outlook for 2017,Nongye Zhanwang(Agricultural Outlook),13(9):57-61(杨宗辉,于群,刘合光,2017,近年马铃薯产业形势与2017年展望,农业展望,13(9):57-61)
Zhang F.,Yin J.L.,Guo Y.Q.,and Yue Y.L.,2018,Research advances on WRKY transcription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),34(1):40-48(张凡,尹俊龙,郭瑛琪,岳艳玲,2018,WRKY转录因子的研究进展,生物技术通报,34(1):40-48)
Zhang Y.,and Wang L.,2005,The WRKY transcription factor superfamily:its origin in eukaryotes and expansion in plants,BMC Evol.Biol.,5(1):1-12
Eulgem T.,Rushton P.J.,Robatzek S.,and Somssich I.E.,2000,The WRKY superfamily of plant transcription factors,Trends Plant Sci.,5(5):199-206
Huang S.X.,and Liu Y.S.,2013,Genome-wide analysis of WRKY transcription factors in solanum tuberosum,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Ap plied&Environmental Biology),19(2):205-214(黄胜雄,刘永胜,2013,土豆WRKY转录因子家族的生物信息学分析,应用与环境生物学报,19(2):205-214)
Joshi R.,Wani S.H.,Singh B.,Boohra A.,Dar Z.A.,Lone A.A.,Pareek A.,and Singla-Pareek S.L.,2016,Transcription factors and plants response to drought stress:current understanding and future directions,Front Plant Sci.,7:1029
Li R.,and Lou Y.G.,2011,Research advances on stress responsive WRKY transcription factors in plants,Shengtai Xuebao(Acta Ecologica Sinica),31(11):3223-3231(李冉,娄永根,2011,植物中逆境反应相关的WRKY转录因子研究进展,生态学报,31(11):3223-3231)
Li X.,Chen Y.F.,and Yang X.,2017,Research progress of WRKY transcription factors in solanaceae plants,Yuanyi Xuebao(Acta Horticulturae Sinica),44(1):170-178(李笑,成玉富,杨旭,2017,茄科植物WRKY转录因子的研究进展,园艺学报,44(1):170-178)
Maeo K.,Hayashi S.,Kojima-Suzuki H.,Kojima-Suzuki H.,Morikami A.,and Nakamura K.,2001,Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins,Biosci.Biotechnol.Biochem.,65(11):2428-2436
Ren Y.J.,Wang H.X.,Li Y.J.,Chen Y.L.,Wang J.,and Tian Z.D.,2015,An earlyβ-aminobutyric acid responsive gene St WRKY5 confers resistance to late blight in potato,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(6):1207-1213(任亚娟,王海霞,李亚军,陈艳林,王静,田振东,2015,β-氨基丁酸早期诱导表达基因St WRKY5参与马铃薯晚疫病抗性,分子植物育种,13(6):1207-1213)
Wang T.,Ma S.,Sun J.Y.,and Ru J.R.,2017,Research progress in physiological race of Phytophthora infestans in China,Zhongguo Malingshu(Chinese Potato Journal),31(1):45-53(王腾,马爽,孙继英,汝甲荣,2017,中国马铃薯晚疫病菌生理小种研究进展,中国马铃薯,31(1):45-53)
Wen F.,Wu X.Z.,Liao L.,Liu X.S.,and Li P.,2017,Research advances on physiological function of WRKY transcription factor in plant stress resistance,Guangxi Zhiwu(Guihaia),7(1):69-79(文锋,吴小祝,廖亮,刘新圣,李鹏,2017,WRKY转录因子在植物抗逆生理中的研究进展,广西植物,7(1):69-79)
Xie Z.,Zhang Z.L.,Zou X.L.,Huang J.,Ruas P.,Thompson D.,and Shen Q.J.,2005,Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells,Plant Physiol.,137(1):176-189
Yang Z.H.,Yu Q.,and Liu H.G.,2017,Situation of potato industry in recent years and its outlook for 2017,Nongye Zhanwang(Agricultural Outlook),13(9):57-61(杨宗辉,于群,刘合光,2017,近年马铃薯产业形势与2017年展望,农业展望,13(9):57-61)
Zhang F.,Yin J.L.,Guo Y.Q.,and Yue Y.L.,2018,Research advances on WRKY transcription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),34(1):40-48(张凡,尹俊龙,郭瑛琪,岳艳玲,2018,WRKY转录因子的研究进展,生物技术通报,34(1):40-48)
Zhang Y.,and Wang L.,2005,The WRKY transcription factor superfamily:its origin in eukaryotes and expansion in plants,BMC Evol.Biol.,5(1):1-12