黄瓜苯丙氨酸解氨酶基因CsPAL的克隆及响应白粉菌侵染的表达分析
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摘要
为了分析苯丙氨酸解氨酶基因(CsPAL)在黄瓜侵染白粉菌中的转录应答响应,对CsPAL基因(GenBank No.JN 675927)及其启动子进行了克隆与序列分析,再利用qRT-PCR技术分析了该基因在黄瓜高抗白粉病品种‘Jin5-508’接种白粉菌后不同时间的相对表达量。结果表明:CsPAL基因全长2 142 bp,编码713个氨基酸;推测CsPAL蛋白存在于细胞质中;系统发育进化树分析发现与拟南芥基因进化距离较近;启动子克隆及序列分析显示该基因能够响应真菌的侵染;组织特异性表达发现CsPAL在在叶片中的表达量最高;qRTPCR结果表明‘:Jin5-508’叶片在接菌16 h内,CsPAL基因的相对表达量于对照间无显著差异;接菌16 h后,CsPAL基因表达量迅速上升,显著高于对照,并在24 h达到最大值;接菌48 h后,CsPAL基因的表达量逐渐下降,但在96 h内仍显著高于相应对照的表达量。综上所述,黄瓜CsPAL基因为白粉菌侵染响应基因,可能与黄瓜对白粉病的抗性具有一定的相关性。
To analyze the role of Cucumis sativus Phenylalanine ammonia-lyase(CsPAL) gene in responding of powdery mildew infection, we cloned and sequenced the CsPAL gene(GenBank No.JN675927) and its promoter, then analyzed the relative expression levels of the gene in the breed of high resistance to powdery mildew 'Jin5-508'at different time-points after inoculation of powdery mildew(PM) pathogen. The results showed that the full length of CsPAL was 2 142 bp, encodes 713 amino acids and located in cytoplasm. Phylogenetic tree analysis showed that the gene was evolutionary close to Arabidopsis PAL gene. Cloning and sequences analysis of the promoter showed that CsPAL gene was able to respond to fungal infection. Tissue-special expression showed that the gene was highly expressed in leaves; qRT-PCR results showed that: there was no significant different in CsPAL expression within 16 h of PM inoculation and control. The relative expression of CsPAL was significantly higher in PM inoculated leaves than those in the control 16 h after treatment and reached the maximum at 24 h. The relative expression of CsPAL was down-regulated gradually after 48 h of treatment, but still significantly higher than control within 96 h. In conclusion, the cloned cucumber CsPAL might be a PM inoculation responsive gene, and might correlate with cucumber PM resistance.
To analyze the role of Cucumis sativus Phenylalanine ammonia-lyase(CsPAL) gene in responding of powdery mildew infection, we cloned and sequenced the CsPAL gene(GenBank No.JN675927) and its promoter, then analyzed the relative expression levels of the gene in the breed of high resistance to powdery mildew 'Jin5-508'at different time-points after inoculation of powdery mildew(PM) pathogen. The results showed that the full length of CsPAL was 2 142 bp, encodes 713 amino acids and located in cytoplasm. Phylogenetic tree analysis showed that the gene was evolutionary close to Arabidopsis PAL gene. Cloning and sequences analysis of the promoter showed that CsPAL gene was able to respond to fungal infection. Tissue-special expression showed that the gene was highly expressed in leaves; qRT-PCR results showed that: there was no significant different in CsPAL expression within 16 h of PM inoculation and control. The relative expression of CsPAL was significantly higher in PM inoculated leaves than those in the control 16 h after treatment and reached the maximum at 24 h. The relative expression of CsPAL was down-regulated gradually after 48 h of treatment, but still significantly higher than control within 96 h. In conclusion, the cloned cucumber CsPAL might be a PM inoculation responsive gene, and might correlate with cucumber PM resistance.
引文
Bi Y.F.,Guo J.,Xu B.H.,Zhang Y.B.,Yi H.P.,Qian C.T.,and Chen J.F.,2015,Resistance evaluation of melon(Cucumis melo L.)to gummy stem blight and expression of PAL gene,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia S-inica),35(2):239-244(毕研飞,郭静,徐兵划,张永兵,伊鸿平,钱春桃,陈劲枫,2015,甜瓜蔓枯病抗性鉴定及PAL基因表达分析,西北植物学报,35(2):239-244)
Chen Y.,Li F.,Tian L.,Huang M.,Deng R.,Li X.,Chen W.,Wu P.,Li M.,Jiang H.,and Wu G.,2017,The phenylalanine ammonia lyase gene LJPAL 1 is involved in plant defense responses to pathogens and plays diverse roles in Lotus japonicus-rhizobium symbioses,Mol.Plant Microbe Interact.,30(9):739-753
He X.,Li Y.,Pandey S.,Yandell B.,Pathak M.,and Weng Y.,2013,QTL mapping of powdery mildew resistance in WI 2 757 cucumber(Cucumis sativus L.),Theor.Appl.Genet.,126(8):2149-2161
Irisarri P.,Zhebentyayeva T.,Errea P.,and Pina A.,2016,Differential expression of phenylalanine ammonia lyase(PAL)genes implies distinct roles in development of graft incompatibility symptoms in Prunus,Sci.Hortic.,204:16-24
Lin X.J.,Xu X.W.,Qian H.M.,Qi X.H.,Xu Q.,and Chen X.H.,2012,Analysis of cucumber chromosome segment introgression line with powdery mildew resistance based on SSRmarkers,Yuanyi Xuebao(Acta Horticulturae Sinica),39(3):485-492(林肖剑,许学文,钱红梅,齐晓花,徐强,陈学好,2012,黄瓜抗白粉病染色体片段导入系的SSR鉴定,园艺学报,39(3):485-492)
Liu R.,Xu S.,Li J.,Hu Y.,and Lin Z.,2006,Expression profile of a PAL gene from Astragalus membranaceus var.Mongholicus and its crucial role in flux into flavonoid biosynthesis.,Plant Cell Rep.,25(7):705-710
MrázováA.,Belay S.A.,Eliá觢ováA.,Perez-Delgado C.M.,KaducováM.,Betti M.,Vega J.M.,and Palove-Balang P.,2017,Expression,activity of phenylalanine-ammonia-lyase and accumulation of phenolic compounds in Lotus japonicus under salt stress,Biologia,72(1):36-42
Nie J.T.,He H.,Peng J.L.,Yang X.Q.,Bie B.B.,Zhao J.L.,Wang Y.L.,Si L.T.,Pan J.S.,and Cai R.,2015,Identification and fine mapping of pm 5.1:a recessive gene for powdery mildew resistance in cucumber(Cucumis sativus L.),Mol.Breeding,35(1):7
Qi X.H.,Xu Q.,Shen L.P.,Alfandi M.,Luo J.J.,and Chen X.H.,2010,Identification of differentially expressed genes between powdery mildew resistant near-isogenic line and susceptible line of cucumber by suppression subtractive hybridization,Sci.Hortic.,126(1):27-32
Rookes J.E.,and Cahill D.M.,2003,A PAL 1 gene promoter-green fluorescent protein reporter system to analyse defence responses in live cells of Arabidopsis thaliana,European Journal of Plant Pathology,109(1):83-94
Tonnessen B.W.,Manosalva P.,Lang J.M.,Baraoidan M.,Bordeos A.,Mauleon R.,Oard J.,Hulbert S.,Leung H.,and Leach J.E.,2015,Rice phenylalanine ammonia-lyase gene OsPAL 4 is associated with broad spectrum disease resistance,Plant Mol.Biol.,87(3):273-286
Wang X.,Wang L.H.,Shi Y.X.,Mao S.L.,Li B.J.,and Zhang B.X.,2009,Changes of three enzymes activities in resistance to pepper Leveillula taurica,Zhongguo Shucai(China Vegetable),1(22):53-57(王萱,王立浩,石延霞,毛胜利,李宝聚,张宝玺,2009,白粉菌侵染过程中不同抗性辣椒品种叶片3种酶活性变化,中国蔬菜,1(22):53-57)
Xu X.,Yu T.,Xu R.,Shi Y.,Lin X.,Xu Q.,Qi X.,and Chen X.,2016,Fine mapping of a dominantly inherited powdery mildew resistance major-effect QTL,Pm1.1,in cucumber identifies a 41.1 kb region containing two tandemly arrayed cysteine-rich receptor-like protein kinase genes.,Theor.Appl.Genet.,129(3):507-516
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Yu G.H.,Wang G.P.,Wang L.,Yi H.Y.,Mou J.J.,Wu H.Y.,Guo X.X.,Wang H.W.,Kong L.R.,and Li A.F.,2015,Cloning of PAL genes and their response to FHB in wheat,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Ressource),16(5):1055-1061(虞光辉,王桂平,王亮,尹华燕,牟晶晶,吴洪燕,郭秀秀,王宏伟,孔令让,李安飞,2015,小麦PAL基因的克隆及赤霉菌诱导下的表达分析,植物遗传资源学报,16(5):1055-1061)
Chen Y.,Li F.,Tian L.,Huang M.,Deng R.,Li X.,Chen W.,Wu P.,Li M.,Jiang H.,and Wu G.,2017,The phenylalanine ammonia lyase gene LJPAL 1 is involved in plant defense responses to pathogens and plays diverse roles in Lotus japonicus-rhizobium symbioses,Mol.Plant Microbe Interact.,30(9):739-753
He X.,Li Y.,Pandey S.,Yandell B.,Pathak M.,and Weng Y.,2013,QTL mapping of powdery mildew resistance in WI 2 757 cucumber(Cucumis sativus L.),Theor.Appl.Genet.,126(8):2149-2161
Irisarri P.,Zhebentyayeva T.,Errea P.,and Pina A.,2016,Differential expression of phenylalanine ammonia lyase(PAL)genes implies distinct roles in development of graft incompatibility symptoms in Prunus,Sci.Hortic.,204:16-24
Lin X.J.,Xu X.W.,Qian H.M.,Qi X.H.,Xu Q.,and Chen X.H.,2012,Analysis of cucumber chromosome segment introgression line with powdery mildew resistance based on SSRmarkers,Yuanyi Xuebao(Acta Horticulturae Sinica),39(3):485-492(林肖剑,许学文,钱红梅,齐晓花,徐强,陈学好,2012,黄瓜抗白粉病染色体片段导入系的SSR鉴定,园艺学报,39(3):485-492)
Liu R.,Xu S.,Li J.,Hu Y.,and Lin Z.,2006,Expression profile of a PAL gene from Astragalus membranaceus var.Mongholicus and its crucial role in flux into flavonoid biosynthesis.,Plant Cell Rep.,25(7):705-710
MrázováA.,Belay S.A.,Eliá觢ováA.,Perez-Delgado C.M.,KaducováM.,Betti M.,Vega J.M.,and Palove-Balang P.,2017,Expression,activity of phenylalanine-ammonia-lyase and accumulation of phenolic compounds in Lotus japonicus under salt stress,Biologia,72(1):36-42
Nie J.T.,He H.,Peng J.L.,Yang X.Q.,Bie B.B.,Zhao J.L.,Wang Y.L.,Si L.T.,Pan J.S.,and Cai R.,2015,Identification and fine mapping of pm 5.1:a recessive gene for powdery mildew resistance in cucumber(Cucumis sativus L.),Mol.Breeding,35(1):7
Qi X.H.,Xu Q.,Shen L.P.,Alfandi M.,Luo J.J.,and Chen X.H.,2010,Identification of differentially expressed genes between powdery mildew resistant near-isogenic line and susceptible line of cucumber by suppression subtractive hybridization,Sci.Hortic.,126(1):27-32
Rookes J.E.,and Cahill D.M.,2003,A PAL 1 gene promoter-green fluorescent protein reporter system to analyse defence responses in live cells of Arabidopsis thaliana,European Journal of Plant Pathology,109(1):83-94
Tonnessen B.W.,Manosalva P.,Lang J.M.,Baraoidan M.,Bordeos A.,Mauleon R.,Oard J.,Hulbert S.,Leung H.,and Leach J.E.,2015,Rice phenylalanine ammonia-lyase gene OsPAL 4 is associated with broad spectrum disease resistance,Plant Mol.Biol.,87(3):273-286
Wang X.,Wang L.H.,Shi Y.X.,Mao S.L.,Li B.J.,and Zhang B.X.,2009,Changes of three enzymes activities in resistance to pepper Leveillula taurica,Zhongguo Shucai(China Vegetable),1(22):53-57(王萱,王立浩,石延霞,毛胜利,李宝聚,张宝玺,2009,白粉菌侵染过程中不同抗性辣椒品种叶片3种酶活性变化,中国蔬菜,1(22):53-57)
Xu X.,Yu T.,Xu R.,Shi Y.,Lin X.,Xu Q.,Qi X.,and Chen X.,2016,Fine mapping of a dominantly inherited powdery mildew resistance major-effect QTL,Pm1.1,in cucumber identifies a 41.1 kb region containing two tandemly arrayed cysteine-rich receptor-like protein kinase genes.,Theor.Appl.Genet.,129(3):507-516
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Yu G.H.,Wang G.P.,Wang L.,Yi H.Y.,Mou J.J.,Wu H.Y.,Guo X.X.,Wang H.W.,Kong L.R.,and Li A.F.,2015,Cloning of PAL genes and their response to FHB in wheat,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Ressource),16(5):1055-1061(虞光辉,王桂平,王亮,尹华燕,牟晶晶,吴洪燕,郭秀秀,王宏伟,孔令让,李安飞,2015,小麦PAL基因的克隆及赤霉菌诱导下的表达分析,植物遗传资源学报,16(5):1055-1061)