橡胶树感白粉病基因LFG1和LFG2的克隆及表达分析
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摘要
白粉病是橡胶树的主要病害之一,为阐明橡胶树与白粉菌分子互作机制,本研究基于已报道的负调控基因LFG为基础,克隆出橡胶树中同源基因LFG1和LFG2,采用同源克隆方法,以橡胶树基因组DNA和cDNA为模板,PCR扩增HbLFG1和HbLFG2基因;利用生物信息学方法分析该基因及其同源蛋白;并分别对白粉菌侵染不同时间和不同病害等级的橡胶树叶片进行RT-PCR测定,检测HbLFG1和HbLFG2的基因表达量。HbLFG1和HbLFG2基因的核酸和氨基酸分析结果分别为:cDNA序列全长均为729 bp及由242个氨基酸编码组成,蛋白的分子量为27.220 kD和27.248 kD,等电点为8.24和8.63,正负电荷残基数为15和12与13和11,脂肪系数是132.52和130.50,不稳定系数为24.19和26.51,总平均亲水性0.958和1.011。两个基因都有7个跨膜结构域,不含有信号肽,蛋白都属于BI-1家族,蛋白模序有5处差异,两个基因与辣椒和柑橘的BI1-like蛋白同源性最为接近。HbLFG1随白粉菌侵染时间和病害等级的增加均呈现先上升后下降的趋势;HbLFG2随病害等级的上升呈现先上升后下降的趋势,但是随白粉菌侵染时间的增加呈现持续上升的趋势。HbLFG1和HbLFG2两个基因的表达量都与橡胶树白粉菌的侵染有关,但两者存在一定差异。本研究有助于深入阐释橡胶树与白粉菌分子互作机制。
Oidium heveae is one of the main diseases of rubber trees. To clarify the interaction mechanism of the rubber tree and the Oidium heveae. It based on the research of the reported negative regulation gene LFG. The study cloned the homologous genes LFG1 and LFG2 in rubber trees, which laid a foundation for elucidating the molecular interaction mechanism between rubber trees and powdery mildew. HbLFG1 and HbLFG2 genes were amplified by homologous cloning method using genomic DNA and cDNA of rubber tree as template. The gene and its homologous protein were analyzed by bioinformatics method. The rubber tree leaves of different disease grades were subjected to RT-PCR to detect the gene expression levels of HbLFG1 and HbLFG2. The results of nucleic acid and amino acid analysis of HbLFG1 and HbLFG2 genes were: The full length of the cDNA sequence was729 bp and 242 amino acids. The molecular weight of the protein was 27.220 kD and 27.248 kD. The isoelectric point was 8.24 and 8.63, the number of positive and negative charge residues is 15 and 12 and 13 and 11, the fat coefficient is 132.52 and 130.50, the instability coefficient is 24.19 and 26.51, the total average hydrophilicity of0.958 and 1.011. Both genes have seven transmembrane domains, do not contain signal peptides, proteins belong to the BI-1 family, and there are five differences in protein motifs. The two genes are closest to the BI1-like proteins of pepper and citrus. HbLFG1 increased first and then decreased with the increase of infection time and disease grade. HbLFG2 showed a trend of increasing first and then decreasing with the increase of disease grade,but it continued to increase with the increase of infection time of powdery mildew. The expression levels of both HbLFG1 and HbLFG2 genes are related to the infection of powdery mildew of rubber tree, but there are some differences between them. These will help to explain the molecular interaction mechanism between rubber tree and powdery mildew.
Oidium heveae is one of the main diseases of rubber trees. To clarify the interaction mechanism of the rubber tree and the Oidium heveae. It based on the research of the reported negative regulation gene LFG. The study cloned the homologous genes LFG1 and LFG2 in rubber trees, which laid a foundation for elucidating the molecular interaction mechanism between rubber trees and powdery mildew. HbLFG1 and HbLFG2 genes were amplified by homologous cloning method using genomic DNA and cDNA of rubber tree as template. The gene and its homologous protein were analyzed by bioinformatics method. The rubber tree leaves of different disease grades were subjected to RT-PCR to detect the gene expression levels of HbLFG1 and HbLFG2. The results of nucleic acid and amino acid analysis of HbLFG1 and HbLFG2 genes were: The full length of the cDNA sequence was729 bp and 242 amino acids. The molecular weight of the protein was 27.220 kD and 27.248 kD. The isoelectric point was 8.24 and 8.63, the number of positive and negative charge residues is 15 and 12 and 13 and 11, the fat coefficient is 132.52 and 130.50, the instability coefficient is 24.19 and 26.51, the total average hydrophilicity of0.958 and 1.011. Both genes have seven transmembrane domains, do not contain signal peptides, proteins belong to the BI-1 family, and there are five differences in protein motifs. The two genes are closest to the BI1-like proteins of pepper and citrus. HbLFG1 increased first and then decreased with the increase of infection time and disease grade. HbLFG2 showed a trend of increasing first and then decreasing with the increase of disease grade,but it continued to increase with the increase of infection time of powdery mildew. The expression levels of both HbLFG1 and HbLFG2 genes are related to the infection of powdery mildew of rubber tree, but there are some differences between them. These will help to explain the molecular interaction mechanism between rubber tree and powdery mildew.
引文
Babaeizad V.,Imani J.,Kogel K.H.,Eichmann R.,and Hückelhoven R.,2009,Over-expression of the cell death regulator bax inhibitor-1 in barley confers reduced or enhanced susceptibility to distinct fungal pathogens,Theor.Appl.Genet.118(3):455-463
De Mattia F.,Gubser C.,Van Dommelen M.M.,Visch H.J.,Distelmaier F.,Postigo A.,Luyten T.,Parys J.B.,De Smedt H.Smith G.L.,Willems P.H.,a nd Van Kuppeveld F.J.,2009Human golgi antiapoptotic protein modulates intracellular calcium fluxes,Mol.Biol.Cell,20(16):3638-3645
Gubser C.,Bergamaschi D.,Hollinshead M.,Lu X.,Van Kuppeveld F.J.M.,and Smith G.L.,20 07,A new inhibitor of apoptosis from vaccinia virus and eukaryotes,PLoS Pathog.3(2):e17
Han D.J.,Li Z.W.,Cao L.,and Chen Y.F.,2003,Advances in Mlo gene resistant to powdery mildew in barley,Xibei Zhiwu Xuebao(Acta Botanica Boreali-occidentalia Sinica),23(3):496-502(韩德俊,李振岐,曹莉,陈耀锋,2003,大麦抗白粉病基因Mlo的研究进展,西北植物学报,23(3):496-502)
Hu L.,Smith T.F.,and Goldberger G.,2009,LFG:a candidate apoptosis regulatory gene family,Apoptosis,14(11):1255-1265
Hückelhoven R.,2004,Bax inhibitor-1,an ancient cell death suppressor in animals and plants with prokaryotic relatives Apoptosis,9(3):299-307
Hückelhoven R.,Dechert C.,and Kogel K.H.,2003,Overexpression of barley bax inhibitor 1 induces breakdown of mlo-mediated penetration resistance to Blumeria graminis,Proc.Natl.Acad.Sci.USA,100(9):5555-5560
Hurtado de Mendoza T.,Perez-Garcia C.G.,Kroll T.T.,Hoong N.H.,O'Leary D.D.,and Verma I.M.,2011,Antiapoptotic protein lifeguard is required for survival and maintenance of purkinje and granular cells,Proc.Natl.Acad.Sci.USA,108(41):17189-17194
Ishikawa T.,Watanabe N.,Nagano M.,Kawai-Yamada M.,and Lam E.,2011,Bax inhibitor-1:a highly conserved endoplasmic reticulum-resident cell death suppressor,Cell Death Differ.,18(8):1271-1278
Li X.,Bin Z.H.,Di R.,Liang P.,He Q.G.,Liu W.B.,Miao W.G.,and Zheng F.C.,2016,Identification of powdery mildew responsive genes in Hevea brasiliensis through m RNA differential display,Int.J.Mol.Sci.,17(2):181
Reimers K.,Choi C.Y.,Bucan V.,and Vogt P.M.,2008,The bax inhibitor-1(BI-1)family in apoptosis and tumorigenesis,Cur.Mol.Med.,8(2):148-156
Reimers K.,Choi C.Y.,Mau-Thek E.,and Vogt P.M.,2006,Sequence analysis shows that lifeguard belongs to a new evolutionarily conserved cytoprotective family,Int.J.Mol.Med.,18(4):729-734
Robinson K.S.,Clements A.,Williams A.C.,Berger C.N.,and Frankel G.,2011,Bax inhibitor 1 in apoptosis and disease,Oncogene,30(21):2391-2400
Rojas-Rivera D.,Armisén R.,Colombo A.,Martínez G.,Eguiguren A.L.,Díaz A.,Kiviluoto S.,Rodríguez D.,Patron M.,Rizzuto R.,Bultynck G.,Concha M.L.,Sierralta J.,Stutzin A.,and Hetz C.,2012,TMBIM3/GRINA is a novel unfolded protein response(UPR)target gene that controls apoptosis through the modulation of ER calcium homeostasis,Cell Death Differ.,19(6):1013-1026
Shukla S.,Fujita K.,Xiao Q.,Liao Z.,Garfield S.,and Srinivasula S.M.,2011,A shear stress responsive gene product PP1201 protects against fas-mediated apoptosis by reducing fas expression on the cell surface,Apoptosis,16(2):162-173
Somia N.V.,Schmitt M.J.,Vetter D.E.,Van Antwerp D.,Heinemann S.F.,and Verma I.M.,1999,LFG:an anti-apoptotic gene that provides protection from fas-mediated cell death,Proc.Natl.Acad Sci.USA,96(22):12667-12672
Wan S.L.,Liang P.,Liu W.B.,Zhang Y.,Miao W.G.,and Zheng F.C.,2014,Cytological analysis of compatible interactions between rubber tree and Oidium heveae,Zhiwu Baohu(Plant Protection),40(3):26-36(万三连,梁鹏,刘文波,张宇,缪卫国,郑服丛,2014,橡胶树与白粉病菌Oidium heveae亲和互作组织细胞学研究,植物保护,40(3):26-36)
Weis C.,Hückelhoven R.,and Eichmann R.,2013,Lifeguard proteins support plant colonization by biotrophic powdery mildew fungi,J.Exp.Bot.,64(12):3855-3867
De Mattia F.,Gubser C.,Van Dommelen M.M.,Visch H.J.,Distelmaier F.,Postigo A.,Luyten T.,Parys J.B.,De Smedt H.Smith G.L.,Willems P.H.,a nd Van Kuppeveld F.J.,2009Human golgi antiapoptotic protein modulates intracellular calcium fluxes,Mol.Biol.Cell,20(16):3638-3645
Gubser C.,Bergamaschi D.,Hollinshead M.,Lu X.,Van Kuppeveld F.J.M.,and Smith G.L.,20 07,A new inhibitor of apoptosis from vaccinia virus and eukaryotes,PLoS Pathog.3(2):e17
Han D.J.,Li Z.W.,Cao L.,and Chen Y.F.,2003,Advances in Mlo gene resistant to powdery mildew in barley,Xibei Zhiwu Xuebao(Acta Botanica Boreali-occidentalia Sinica),23(3):496-502(韩德俊,李振岐,曹莉,陈耀锋,2003,大麦抗白粉病基因Mlo的研究进展,西北植物学报,23(3):496-502)
Hu L.,Smith T.F.,and Goldberger G.,2009,LFG:a candidate apoptosis regulatory gene family,Apoptosis,14(11):1255-1265
Hückelhoven R.,2004,Bax inhibitor-1,an ancient cell death suppressor in animals and plants with prokaryotic relatives Apoptosis,9(3):299-307
Hückelhoven R.,Dechert C.,and Kogel K.H.,2003,Overexpression of barley bax inhibitor 1 induces breakdown of mlo-mediated penetration resistance to Blumeria graminis,Proc.Natl.Acad.Sci.USA,100(9):5555-5560
Hurtado de Mendoza T.,Perez-Garcia C.G.,Kroll T.T.,Hoong N.H.,O'Leary D.D.,and Verma I.M.,2011,Antiapoptotic protein lifeguard is required for survival and maintenance of purkinje and granular cells,Proc.Natl.Acad.Sci.USA,108(41):17189-17194
Ishikawa T.,Watanabe N.,Nagano M.,Kawai-Yamada M.,and Lam E.,2011,Bax inhibitor-1:a highly conserved endoplasmic reticulum-resident cell death suppressor,Cell Death Differ.,18(8):1271-1278
Li X.,Bin Z.H.,Di R.,Liang P.,He Q.G.,Liu W.B.,Miao W.G.,and Zheng F.C.,2016,Identification of powdery mildew responsive genes in Hevea brasiliensis through m RNA differential display,Int.J.Mol.Sci.,17(2):181
Reimers K.,Choi C.Y.,Bucan V.,and Vogt P.M.,2008,The bax inhibitor-1(BI-1)family in apoptosis and tumorigenesis,Cur.Mol.Med.,8(2):148-156
Reimers K.,Choi C.Y.,Mau-Thek E.,and Vogt P.M.,2006,Sequence analysis shows that lifeguard belongs to a new evolutionarily conserved cytoprotective family,Int.J.Mol.Med.,18(4):729-734
Robinson K.S.,Clements A.,Williams A.C.,Berger C.N.,and Frankel G.,2011,Bax inhibitor 1 in apoptosis and disease,Oncogene,30(21):2391-2400
Rojas-Rivera D.,Armisén R.,Colombo A.,Martínez G.,Eguiguren A.L.,Díaz A.,Kiviluoto S.,Rodríguez D.,Patron M.,Rizzuto R.,Bultynck G.,Concha M.L.,Sierralta J.,Stutzin A.,and Hetz C.,2012,TMBIM3/GRINA is a novel unfolded protein response(UPR)target gene that controls apoptosis through the modulation of ER calcium homeostasis,Cell Death Differ.,19(6):1013-1026
Shukla S.,Fujita K.,Xiao Q.,Liao Z.,Garfield S.,and Srinivasula S.M.,2011,A shear stress responsive gene product PP1201 protects against fas-mediated apoptosis by reducing fas expression on the cell surface,Apoptosis,16(2):162-173
Somia N.V.,Schmitt M.J.,Vetter D.E.,Van Antwerp D.,Heinemann S.F.,and Verma I.M.,1999,LFG:an anti-apoptotic gene that provides protection from fas-mediated cell death,Proc.Natl.Acad Sci.USA,96(22):12667-12672
Wan S.L.,Liang P.,Liu W.B.,Zhang Y.,Miao W.G.,and Zheng F.C.,2014,Cytological analysis of compatible interactions between rubber tree and Oidium heveae,Zhiwu Baohu(Plant Protection),40(3):26-36(万三连,梁鹏,刘文波,张宇,缪卫国,郑服丛,2014,橡胶树与白粉病菌Oidium heveae亲和互作组织细胞学研究,植物保护,40(3):26-36)
Weis C.,Hückelhoven R.,and Eichmann R.,2013,Lifeguard proteins support plant colonization by biotrophic powdery mildew fungi,J.Exp.Bot.,64(12):3855-3867