烟草NtMLO5a和NtMLO5b基因克隆和表达分析
|
摘要
为研究MLO基因在烟草(Nicotiana tabacum)中的特性与功能,本研究采用同源克隆方法从烟草中克隆到NtMLO5a和NtMLO5b两个基因。序列分析表明,烟草NtMLO5a和NtMLO5b基因开放阅读框全长均是1 794 bp,均编码597个氨基酸的蛋白,相对分子质量均为68 kDa,等电点则分别为9.27和9.21。不同物种中MLO蛋白进化分析表明NtMLO5a和NtMLO5b蛋白与辣椒(Capsicum annuum)和野生番茄(Solanum pennellii)的MLO蛋白高度相似。实时荧光定量PCR结果显示,烟草‘K326’的NtMLO5a和NtMLO5b基因在根、茎、叶中均有表达,且在根中的表达水平最高,在花中只检测到NtMLO5b基因表达。白粉病、干旱和低温胁迫均提高了NtMLO5b基因的表达,接种白粉病48 h后和低温处理24 h后, NtMLO5b基因的表达量达到各自的最高值,分别为处理前的2.5倍和13.52倍;干旱处理后, NtMLO5b基因的表达持续上升, 8 d后为处理前的7.54倍。白粉病和干旱对NtMLO5a基因表达量没有明显影响;低温处理12 h后其表达量达到最大值,是处理前的13.72倍。
In order to study the characteristic and fuction of MLO in tobacco(Nicotiana tabacum),we cloned two MLO genes from tobacco‘K326’by homologous cloning technology.NtMLO5a and NtMLO5b both have an 1 794 bp open reading frame(ORF),encoding 597 amino acids.The putative protein molecular weight of NtMLO5a is 68 kDa and its theoretical isoelectric point is 9.27.The putative protein molecular weight and theoretical isoelectric point of NtMLO5b is 68 kDa and 9.21,respectively.Evolution analysis result suggests NtMLO5a and NtMLO5b have the nearest genetic relationship with Capsicum annuum and Solanum pennellii MLOs.Real-time PCR result shows that NtMLO5a and NtMLO5b expressed in the roots,stems,and leaves of tobacco‘K326’,with the highest expression level in roots,but in flower only the transcript of NtMLO5b was detected.NtMLO5b expression can be induced by different stresses.The expression level peaks at 48 h after powdery mildew inoculation and 24 h after low temperature treatment,2.5 and 13.52 folds than the level at 0 h,respectively.While the expression level of NtMLO5b kept on rising during 0–8 d after drought treatment,up to7.54 fold at 8 d than 0 d.Expression level of NtMLO5a showed no significant difference after powdery mildew and drought treatments,but low temperature can induce NtMLO5a expression and the maximum(13.72 fold)appeared at 12 h after treatment.
In order to study the characteristic and fuction of MLO in tobacco(Nicotiana tabacum),we cloned two MLO genes from tobacco‘K326’by homologous cloning technology.NtMLO5a and NtMLO5b both have an 1 794 bp open reading frame(ORF),encoding 597 amino acids.The putative protein molecular weight of NtMLO5a is 68 kDa and its theoretical isoelectric point is 9.27.The putative protein molecular weight and theoretical isoelectric point of NtMLO5b is 68 kDa and 9.21,respectively.Evolution analysis result suggests NtMLO5a and NtMLO5b have the nearest genetic relationship with Capsicum annuum and Solanum pennellii MLOs.Real-time PCR result shows that NtMLO5a and NtMLO5b expressed in the roots,stems,and leaves of tobacco‘K326’,with the highest expression level in roots,but in flower only the transcript of NtMLO5b was detected.NtMLO5b expression can be induced by different stresses.The expression level peaks at 48 h after powdery mildew inoculation and 24 h after low temperature treatment,2.5 and 13.52 folds than the level at 0 h,respectively.While the expression level of NtMLO5b kept on rising during 0–8 d after drought treatment,up to7.54 fold at 8 d than 0 d.Expression level of NtMLO5a showed no significant difference after powdery mildew and drought treatments,but low temperature can induce NtMLO5a expression and the maximum(13.72 fold)appeared at 12 h after treatment.
引文
Appiano M,Pavan S,Catalano D,et al(2015).Identification of candidate MLO powdery mildew susceptibility genes in cultivated Solanaceae and functional characterization of tobacco NtMLO1.Transgenic Res,24(5):847-858
Bai Y,Pavan S,Zheng Z,et al(2008).Naturally occurring broad-spectrum powdery mildew resistance in a Central American tomato accession is caused by loss of Mlo function.Mol Plant Microbe In,21(1):30-39
Bidzinski P,Noir S,Shahi S,et al(2014).Physiological characterization and genetic modifiers of aberrant root thigmomorphogenesis in mutants of Arabidopsis thaliana MILDEW LOCUS O genes.Plant Cell Environ,37:2738-2753
Büschges R,Hollricher K,Panstruga R,et al(1997).The barley Mlo gene:a novel control element of plant pathogen resistance.Cell,88:695-705
Chen Z,Hartmann HA,Wu MJ,et al(2006).Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins.Plant Mol Biol,60:583-597
Chen Z,Noir S,Kwaaitaal M,et al(2009).Two seven-transmembrane domain MILDEW RESISTANCE LOCUS Oproteins cofunction in Arabidopsis root thigmomorphogenesis.Plant Cell,21:1972-1991
Consonni C,Humphry ME,Hartmann HA,et al(2006).Conserved requirement for a plant host cell protein in powdery mildew pathogenesis.Nat Genet,38:716-720
Fujimura T,Sato S,Tajima T,et al(2016).Powdery mildew resistance in the Japanese domestic tobacco cultivar Kokubu is associated with aberrant splicing of MLO orthologues.Plant Pathol,65:1358-1365
International Barley Genome Sequencing Consortium(2012).A physical,genetic and functional sequence assembly of the barley genome.Nature,491(7426):711-716
Kessler SA,Shimosato-Asano H,Keinath NF,et al(2010).Conserved molecular components for pollen tube reception and fungal invasion.Science,330:968-971
Konishi S,Sasakuma T,Sasanuma T(2010).Identification of novel Mlo family members in wheat and their genetic characterization.Genes Genet Syst,85:167-175
Kusch S,Pesch L,Panstruga R(2016).Comprehensive phylogenetic analysis sheds light on the diversity and origin of the MLO family of integral membrane proteins.Genome Biol Evol,8(3):878-895
Liu Q,Zhu H(2008).Molecular evolution of the MLO gene family in Oryza sativa and their functional divergence.Gene,409:1-10
Panstruga R(2005).Serpentine plant MLO proteins as entry portals for powdery mildew fungi.Biochem Soc Trans,33:389-392
Vogel J,Somerville S(2000).Isolation and characterization of powdery mildew-resistant Arabidopsis mutants.Proc Natl Acad Sci USA,97:1897-1902
Wang X,Ma Q,Dou L,et al(2016).Genome-wide characterization and comparative analysis of the MLO gene family in cotton.Plant Physiol Biochem,103:106-119
Yi J,An S,An G(2014).OsMLO12,encoding seven transmembrane proteins,is involved with pollen hydration in rice.Plant Reprod,27:169-180
Bai Y,Pavan S,Zheng Z,et al(2008).Naturally occurring broad-spectrum powdery mildew resistance in a Central American tomato accession is caused by loss of Mlo function.Mol Plant Microbe In,21(1):30-39
Bidzinski P,Noir S,Shahi S,et al(2014).Physiological characterization and genetic modifiers of aberrant root thigmomorphogenesis in mutants of Arabidopsis thaliana MILDEW LOCUS O genes.Plant Cell Environ,37:2738-2753
Büschges R,Hollricher K,Panstruga R,et al(1997).The barley Mlo gene:a novel control element of plant pathogen resistance.Cell,88:695-705
Chen Z,Hartmann HA,Wu MJ,et al(2006).Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins.Plant Mol Biol,60:583-597
Chen Z,Noir S,Kwaaitaal M,et al(2009).Two seven-transmembrane domain MILDEW RESISTANCE LOCUS Oproteins cofunction in Arabidopsis root thigmomorphogenesis.Plant Cell,21:1972-1991
Consonni C,Humphry ME,Hartmann HA,et al(2006).Conserved requirement for a plant host cell protein in powdery mildew pathogenesis.Nat Genet,38:716-720
Fujimura T,Sato S,Tajima T,et al(2016).Powdery mildew resistance in the Japanese domestic tobacco cultivar Kokubu is associated with aberrant splicing of MLO orthologues.Plant Pathol,65:1358-1365
International Barley Genome Sequencing Consortium(2012).A physical,genetic and functional sequence assembly of the barley genome.Nature,491(7426):711-716
Kessler SA,Shimosato-Asano H,Keinath NF,et al(2010).Conserved molecular components for pollen tube reception and fungal invasion.Science,330:968-971
Konishi S,Sasakuma T,Sasanuma T(2010).Identification of novel Mlo family members in wheat and their genetic characterization.Genes Genet Syst,85:167-175
Kusch S,Pesch L,Panstruga R(2016).Comprehensive phylogenetic analysis sheds light on the diversity and origin of the MLO family of integral membrane proteins.Genome Biol Evol,8(3):878-895
Liu Q,Zhu H(2008).Molecular evolution of the MLO gene family in Oryza sativa and their functional divergence.Gene,409:1-10
Panstruga R(2005).Serpentine plant MLO proteins as entry portals for powdery mildew fungi.Biochem Soc Trans,33:389-392
Vogel J,Somerville S(2000).Isolation and characterization of powdery mildew-resistant Arabidopsis mutants.Proc Natl Acad Sci USA,97:1897-1902
Wang X,Ma Q,Dou L,et al(2016).Genome-wide characterization and comparative analysis of the MLO gene family in cotton.Plant Physiol Biochem,103:106-119
Yi J,An S,An G(2014).OsMLO12,encoding seven transmembrane proteins,is involved with pollen hydration in rice.Plant Reprod,27:169-180