巴西橡胶树(Hevea brasiliensis)HbMlo2基因克隆与表达分析
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摘要
Mlo (mildew resistance locus O)是一种植物防卫相关基因,对植物的抗病性有负向调控作用。本研究采用基因编辑技术等方法为培育橡胶树抗病品种作出铺垫,进一步填补Mlo家族成员。通过对拟南芥的AtMlo2基因在巴西橡胶树上进行比对,根据同源性克隆得到一个cDNA序列,将其命名为HbMlo2。根据生物信息学分析得出,该基因cDNA全长1 725 bp,编码574个氨基酸,该蛋白的分子量为65.78 kD,等电点为8.98,8个跨膜域,没有信号肽,HbMlo2基因与已知的木薯的Mlo基因相似性达到83%。白粉菌侵染橡胶树叶组织后,通过qRT-PCR发现HbMlo2基因显著上调。本研究结果初步表明,HbMlo2可能参与橡胶树的抗病反应机制,为橡胶树抗病育种的研究提供新思路。
Mlo(mildew resistance locus O) is a plant defense-related gene that has a negative regulatory effect on plant disease resistance. This study paves the way for the cultivation of Hevea brasiliensis resistant varieties for methods such as genetic editing techniques, and further filling the Mlo family members. By aligning the AtMlo2 gene of Arabidopsis thaliana on Hevea brasiliensis, a c DNA sequence was cloned according to homology and designated as HbMlo2. Bioinformatics analysis showed that the cDNA of this gene was 1 725 bp in length which encoded 574 amino acids. The molecular weight of this protein was 65.78 kD and the isoelectric point was 8.98, it had 8 transmembrane domains and no signal peptide. Similarity of the known cassava between HbMlo2 gene and the Mlo gene reached 83%. After infestation of Hevea brasiliensis leaf tissue by powdery mildew, the HbMlo2 gene was significantly up-regulated by qRT-PCR. The results of this study preliminarily indicated that HbMlo2 might be involved in the disease resistance reaction mechanism of Hevea brasiliensis, which providing new ideas for the research of Hevea brasiliensis disease resistance breeding.
Mlo(mildew resistance locus O) is a plant defense-related gene that has a negative regulatory effect on plant disease resistance. This study paves the way for the cultivation of Hevea brasiliensis resistant varieties for methods such as genetic editing techniques, and further filling the Mlo family members. By aligning the AtMlo2 gene of Arabidopsis thaliana on Hevea brasiliensis, a c DNA sequence was cloned according to homology and designated as HbMlo2. Bioinformatics analysis showed that the cDNA of this gene was 1 725 bp in length which encoded 574 amino acids. The molecular weight of this protein was 65.78 kD and the isoelectric point was 8.98, it had 8 transmembrane domains and no signal peptide. Similarity of the known cassava between HbMlo2 gene and the Mlo gene reached 83%. After infestation of Hevea brasiliensis leaf tissue by powdery mildew, the HbMlo2 gene was significantly up-regulated by qRT-PCR. The results of this study preliminarily indicated that HbMlo2 might be involved in the disease resistance reaction mechanism of Hevea brasiliensis, which providing new ideas for the research of Hevea brasiliensis disease resistance breeding.
引文
Acevedo-Garcia J.,Kusch S.,and Panstruga R.,2014,Magical mystery tour:MLO proteins in plant immunity and beyond,New Phytol.,204(2):273-281
Bai Y.,Pavan S.,Zheng Z.,Zappel N.F.,Reinstadler A.,Lotti C.,De Giovanni C.,Ricciardi L.,Lindhout P.,Visser R.,Theres K.,and Panstruga R.,2008,Naturally occurring broad-spectrum powdery mildew resistance in a central American tomato accession is caused by loss of mlo function,Mol.Plant Microbe Interact.,21(1):30-39
Buschges R.,Hollricher K.,Panstruga R.,Simons G.,Wolter M.,Frijters A.,Daelen R.V.,Lee T.V.D.,Diergaarde P.,Groenendijk J.,Topsch S.,Vos P.,Salamini F.,and Schulze-Lefert P.,1997,The barley Mlo gene:a novel control element of plant pathogen resistance,Cell,88(5):695-705
Cheng H.,Kong W.P.,He Q.W.,and Wang X.W.,2013,CmM-LO2:a new gene associated with melon powdery mildew,Yuanyi Xuebao(Acta Horticulturae Sinica),40(3):540-548(程鸿,孔维萍,何启伟,王晓巍,2013,CmMLO2:一个与甜瓜白粉病感病相关的新基因,园艺学报,40(3):540-548)
Consonni C.,Humphry M.E.,Hartmann H.A.,Livaja M.,Durner J.,Westphal L.,Vogel J.,Lipka V.,Kemmerling B.,SchulzeLefert P.,Somerville S.C.,and Panstruga R.,2006,Conserved requirement for a plant host cell protein in powdery mildew pathogenesis,Nat.Genet.,38(6):716-720
Deshmukh R.,Singh V.K.,and Singh B.D.,2014,Comparative phylogenetic analysis of genome-wide Mlo gene family members from Glycine max and Arabidopsis thaliana,Mol.Genet.Genomics,289(3):345-359
Devoto A.,Piffanelli P.,Nilsson I.,Wallin E.,Panstruga R.,Heijne G.V.,and SchulzeLefert P.,1999,Topology,subcellular localization,and sequence diversity of the Mlo family in plants,J.Biol.Chem.,274(49):34993-35004
Elliott C.,Zhou F.,Spielmeyer W.,Panstruga R.,and Schulzelefert P.,2002,Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus,Mol.Plant Microbe Interact.,15(10):1069-1077
Guo P.,Liang P.,He Q.G.,Liu W.B.,Lin C.H.,Miao W.G.,and Zheng F.C.,2016,Clone and expression analysis of MAT-related gene(STE2 gene)from Oidium heveae B.A.Steinm,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):879-885(郭鹏,梁鹏,何其光,刘文波,林春花,缪卫国,郑服丛,2016,橡胶树白粉菌MAT相关基因(STE2基因)的克隆及表达分析,基因组学与应用生物学,35(4):879-885)
He H.X.,Zhang Y.,Wang M.,Yang Y.,and Zheng F.C.,2016,Molecular cloning and characterization of HbMlo7 gene in rubber tree(Hevea brasiliensis),Zhiwu Shengli Xuebao(Plant Physiology Journal),52(6):917-925(何海霞,张宇,王萌,杨叶,郑服丛,2016,巴西橡胶树(Hevea brasiliensis)HbMlo7基因克隆与表达分析,植物生理学报,52(6):917-925)
Kim M.C.,Lee S.H.,Kim J.K.,Chun H.J.,Choi M.S.,Chung W.S.,Moon B.C.,Kang C.H.,Park C.Y.,Yoo J.H.,Kang Y.H.,Koo S.C.,Koo Y.D.,Jung J.C.,Kim S.T.,Schulze-Lefert P.,Lee S.Y.,and Cho M.J.,2002,Mlo,a modulator of plant defense and cell death,is a novel calmodulin-binding protein,J.Biol.Chem.,277(22):19304-19314
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Pavan S.,Jacobsen E.,Visser R.G.,and Bai Y.,2010,Loss of susceptibility as a novel breeding s trategy for durable and broad-spectrum resistance,Mol.Breed.,25(1):1-12
Pavan S.,Schiavulli A.,Appiano M.,Marcotrigiano A.R.,Cillo F.,Visser R.G.,Bai Y.,Lotti C.,and Ricciardi L.,2011,Pea powdery mildew er1 resistance is associated to loss-of-function mutations at a MLO homologous locus,Theor.Appl.Genet.,123(8):1425-1431
Qin B.,Wang M.,Xue S.,Hu Z.Y.,Zheng F.C.,and Zhang Y.,2013,Cloning and sequence characterization of a Mlo gene in Hevea brasiliensis Mull.,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),29(31):21-26(覃碧,王萌,薛松,胡中昀,郑服丛,张宇,2013,巴西橡胶树1个Mlo基因克隆及其序列特征分析,中国农学通报,29(31):21-26)
Schweizer P.,Pokorny J.,Schulze-Lefert P.,and Dudler R.,2000,Double-stranded RNA interferes with gene function at the single-cell level in cereals,Plant J.,24(6):895-903
Sun Y.Q.,2015,Cloning and quantitative RT-PCR analysis of pathogenicity genes and MAPK pathway genes of Oidium heveae,Thesis for M.S.,Hainan Univeristy,Supervisor:Zheng F.C.,and Miao W.G.,pp.1-3(孙雅琦,2015,橡胶树白粉菌致病相关基因及MAPK途径相关基因的克隆和定量分析,硕士学位论文,海南大学,导师:郑服丛,缪卫国,pp.1-3)
Wang X.,Ma Q.,Dou L.,Liu Z.,Peng R.,and Yu S.,2016,Genome-wide characterization and comparative analysis of the MLO gene family in cotton,Plant Physiol.Biochem.,103:106-119
Win K.T.,Zhang C.,and Lee S.,2018,Genome wide identifcation and description of MLO family genes in pumpkin(Cucurbita maxima Duch.),Hortic.Environ.Biote.,59(3):397-410
Zeng R.Z.,Duan C.F.,Li X.Y.,Tian W.M.,and Nie Z.Y.,2009,Vacuolar-type in organic pyrophosphatase located on the rubber particlein the latex is an essential enzyme in regulation of the rubberbiosynthesis in Hevea brasiliensis,Plant Sci.,176(5):602-607
Bai Y.,Pavan S.,Zheng Z.,Zappel N.F.,Reinstadler A.,Lotti C.,De Giovanni C.,Ricciardi L.,Lindhout P.,Visser R.,Theres K.,and Panstruga R.,2008,Naturally occurring broad-spectrum powdery mildew resistance in a central American tomato accession is caused by loss of mlo function,Mol.Plant Microbe Interact.,21(1):30-39
Buschges R.,Hollricher K.,Panstruga R.,Simons G.,Wolter M.,Frijters A.,Daelen R.V.,Lee T.V.D.,Diergaarde P.,Groenendijk J.,Topsch S.,Vos P.,Salamini F.,and Schulze-Lefert P.,1997,The barley Mlo gene:a novel control element of plant pathogen resistance,Cell,88(5):695-705
Cheng H.,Kong W.P.,He Q.W.,and Wang X.W.,2013,CmM-LO2:a new gene associated with melon powdery mildew,Yuanyi Xuebao(Acta Horticulturae Sinica),40(3):540-548(程鸿,孔维萍,何启伟,王晓巍,2013,CmMLO2:一个与甜瓜白粉病感病相关的新基因,园艺学报,40(3):540-548)
Consonni C.,Humphry M.E.,Hartmann H.A.,Livaja M.,Durner J.,Westphal L.,Vogel J.,Lipka V.,Kemmerling B.,SchulzeLefert P.,Somerville S.C.,and Panstruga R.,2006,Conserved requirement for a plant host cell protein in powdery mildew pathogenesis,Nat.Genet.,38(6):716-720
Deshmukh R.,Singh V.K.,and Singh B.D.,2014,Comparative phylogenetic analysis of genome-wide Mlo gene family members from Glycine max and Arabidopsis thaliana,Mol.Genet.Genomics,289(3):345-359
Devoto A.,Piffanelli P.,Nilsson I.,Wallin E.,Panstruga R.,Heijne G.V.,and SchulzeLefert P.,1999,Topology,subcellular localization,and sequence diversity of the Mlo family in plants,J.Biol.Chem.,274(49):34993-35004
Elliott C.,Zhou F.,Spielmeyer W.,Panstruga R.,and Schulzelefert P.,2002,Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus,Mol.Plant Microbe Interact.,15(10):1069-1077
Guo P.,Liang P.,He Q.G.,Liu W.B.,Lin C.H.,Miao W.G.,and Zheng F.C.,2016,Clone and expression analysis of MAT-related gene(STE2 gene)from Oidium heveae B.A.Steinm,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):879-885(郭鹏,梁鹏,何其光,刘文波,林春花,缪卫国,郑服丛,2016,橡胶树白粉菌MAT相关基因(STE2基因)的克隆及表达分析,基因组学与应用生物学,35(4):879-885)
He H.X.,Zhang Y.,Wang M.,Yang Y.,and Zheng F.C.,2016,Molecular cloning and characterization of HbMlo7 gene in rubber tree(Hevea brasiliensis),Zhiwu Shengli Xuebao(Plant Physiology Journal),52(6):917-925(何海霞,张宇,王萌,杨叶,郑服丛,2016,巴西橡胶树(Hevea brasiliensis)HbMlo7基因克隆与表达分析,植物生理学报,52(6):917-925)
Kim M.C.,Lee S.H.,Kim J.K.,Chun H.J.,Choi M.S.,Chung W.S.,Moon B.C.,Kang C.H.,Park C.Y.,Yoo J.H.,Kang Y.H.,Koo S.C.,Koo Y.D.,Jung J.C.,Kim S.T.,Schulze-Lefert P.,Lee S.Y.,and Cho M.J.,2002,Mlo,a modulator of plant defense and cell death,is a novel calmodulin-binding protein,J.Biol.Chem.,277(22):19304-19314
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Pavan S.,Jacobsen E.,Visser R.G.,and Bai Y.,2010,Loss of susceptibility as a novel breeding s trategy for durable and broad-spectrum resistance,Mol.Breed.,25(1):1-12
Pavan S.,Schiavulli A.,Appiano M.,Marcotrigiano A.R.,Cillo F.,Visser R.G.,Bai Y.,Lotti C.,and Ricciardi L.,2011,Pea powdery mildew er1 resistance is associated to loss-of-function mutations at a MLO homologous locus,Theor.Appl.Genet.,123(8):1425-1431
Qin B.,Wang M.,Xue S.,Hu Z.Y.,Zheng F.C.,and Zhang Y.,2013,Cloning and sequence characterization of a Mlo gene in Hevea brasiliensis Mull.,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),29(31):21-26(覃碧,王萌,薛松,胡中昀,郑服丛,张宇,2013,巴西橡胶树1个Mlo基因克隆及其序列特征分析,中国农学通报,29(31):21-26)
Schweizer P.,Pokorny J.,Schulze-Lefert P.,and Dudler R.,2000,Double-stranded RNA interferes with gene function at the single-cell level in cereals,Plant J.,24(6):895-903
Sun Y.Q.,2015,Cloning and quantitative RT-PCR analysis of pathogenicity genes and MAPK pathway genes of Oidium heveae,Thesis for M.S.,Hainan Univeristy,Supervisor:Zheng F.C.,and Miao W.G.,pp.1-3(孙雅琦,2015,橡胶树白粉菌致病相关基因及MAPK途径相关基因的克隆和定量分析,硕士学位论文,海南大学,导师:郑服丛,缪卫国,pp.1-3)
Wang X.,Ma Q.,Dou L.,Liu Z.,Peng R.,and Yu S.,2016,Genome-wide characterization and comparative analysis of the MLO gene family in cotton,Plant Physiol.Biochem.,103:106-119
Win K.T.,Zhang C.,and Lee S.,2018,Genome wide identifcation and description of MLO family genes in pumpkin(Cucurbita maxima Duch.),Hortic.Environ.Biote.,59(3):397-410
Zeng R.Z.,Duan C.F.,Li X.Y.,Tian W.M.,and Nie Z.Y.,2009,Vacuolar-type in organic pyrophosphatase located on the rubber particlein the latex is an essential enzyme in regulation of the rubberbiosynthesis in Hevea brasiliensis,Plant Sci.,176(5):602-607