不结球白菜抗病相关基因BcSGT1的克隆及表达分析
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摘要
SGT1(suppressor of the G2 allele of skp1)基因作为植物抗病信号途径中的重要元件,广泛参与植物细胞周期、逆境反应和信号转导等过程。为研究不结球白菜(Brassica rapa ssp. chinensis)抗病相关基因BcSGT1的结构和表达特征,本研究通过RACE (rapid-amplification of cDNA ends)技术,以抗病品种‘苏州青’叶片为材料克隆到BcSGT1基因的全长c DNA序列;采用q RT-PCR分析该基因在霜霉病菌(Peronospora parasitica)及黑斑病菌(Alternaria brassicicola)诱导处理条件下的表达模式;用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)技术分析该基因的原核表达特征。序列分析结果表明,BcSGT1基因的c DNA全长为1 418 bp,其中开放阅读框长度为1 074 bp,共编码358个氨基酸(GenBank No. AB495003)。氨基酸同源性的系统进化分析表明,不结球白菜BcSGT1基因与同科植物的进化关系相近,其中与大白菜(Brassica rapa ssp. pekinensis)第3号染色体的基因(Bra000741)同源性最高(97%)。qRT-PCR分析表明,霜霉病菌侵染处理下,BcSGT1基因在抗病品种‘苏州青’和感病品种‘矮脚黄’中的表达量分别于24、48 h达到峰值,且在‘苏州青’中的表达量峰值约为‘矮脚黄’中的2.1倍(P<0.01);黑斑病菌侵染处理下,BcSGT1基因在抗病品种‘苏州青’和感病品种‘矮脚黄’中的表达量分别于12、24 h达到峰值,且在‘苏州青’中的表达量峰值约为‘矮脚黄’中的2.0倍(P<0.01);霜霉病菌侵染处理24和48 h后,黑斑病菌侵染处理12、24和48 h后,BcSGT1基因在抗病品种‘苏州青’中的表达量均显著高于感病品种‘矮脚黄’中的表达量(P<0.01)。原核表达载体经异丙基-β-D-硫代半乳糖苷(isopropylβ-D-thiogalactoside, IPTG)诱导表达出相对分子质量约为39 kD的融合蛋白。BcSGT1基因是不结球白菜霜霉病菌及黑斑病菌抗病反应的重要组成部分,且本研究成功将BcSGT1融合表达进大肠杆菌(Escherichia coli)中。本研究为高产、优质及抗病不结球白菜新品种的选育提供重要的理论依据。
SGT1(suppressor of the G2 allele of skp1) as an important element for plant disease resistance, isinvolved widely in plant cell cycling, stress response, protein ubiquitination and signal transduction processes,and it plays an important role in plant disease resistance. In order to study the structure and expression characteristics of the disease-related gene BcSGT1 in non-heading Chinese cabbage(Brassica rapa ssp.chinensis), the full-length c DNA sequence of BcSGT1 gene was cloned from the resistant variety 'Suzhouqing'by RACE technique. The expression analysis of gene was used by qRT-PCR. The expression pattern of Peronospora parasitica and Alternaria brassicicola induced treatment conditions, sodium dodecyl sulphatepolyacrylamide gel electrophoresis(SDS-PAGE) technique was used to analyze the prokaryotic expression characteristics for the gene. The analysis results of sequence indicated that the full-length cDNA of BcSGT1 gene was 1 418 bp, and the open reading frame was 1 074 bp in length, encoding a total of 358 amino acids.The relative molecular weight was 39.77 kD, and the protein theoretical isoelectric point was 5.05(GenBank No. AB495003). The evolutionary analysis of amino acid homologous system showed that the BcSGT1 gene of the non-heading Chinese cabbage had similar evolutionary relationship with the same family plant, and the highest homology(97%) was found with the chromosome 3 gene of Brassica rapa(Bra000741). The qRT-PCR analysis showed that under the infection of P. parasitica, the expression level of BcSGT1 gene in the resistant variety 'Suzhouqing' peaked at the 24 h, while the expression level in the susceptible variety 'Aijiaohuang'reached the peak at the 48 h. The peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.1 times higher than that in the susceptible variety 'Aijiaohuang'(P<0.01). With the infection of A.brassicicola, the expression level of BcSGT1 in 'Suzhouqing' reached its peak at 12 h, while the expression level in the susceptible variety 'Aijiaohuang' peaked at 24 h, and the peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.0 times higher than the peak expression in the susceptible variety'Aijiaohuang'(P<0.01). The expression level of BcSGT1 in 'Suzhouqing' is significantly higher than the expression level in 'Aijiaohuang' after 24 and 48 hours of infection by P. parasitica, and 12 h, 24 h, 48 h infection by A. brassicicola(P<0.01). The prokaryotic expression vector was induced by isopropyl β-Dthiogalactoside(IPTG) to express a fusion protein with a relative molecular mass of about 39 kD. The BcSGT1 gene of non-heading Chinese cabbage played an important role in the infection of P. parasitica and A.brassicicola, and BcSGT1 successfully achieved fusion expression in Escherichia coli, which provided the conditions for further protein level research and transgenic function research. It will also provide an important theoretical value for the selection of high-yield, high-quality and disease-related non-heading Chinese cabbage varieties.
SGT1(suppressor of the G2 allele of skp1) as an important element for plant disease resistance, isinvolved widely in plant cell cycling, stress response, protein ubiquitination and signal transduction processes,and it plays an important role in plant disease resistance. In order to study the structure and expression characteristics of the disease-related gene BcSGT1 in non-heading Chinese cabbage(Brassica rapa ssp.chinensis), the full-length c DNA sequence of BcSGT1 gene was cloned from the resistant variety 'Suzhouqing'by RACE technique. The expression analysis of gene was used by qRT-PCR. The expression pattern of Peronospora parasitica and Alternaria brassicicola induced treatment conditions, sodium dodecyl sulphatepolyacrylamide gel electrophoresis(SDS-PAGE) technique was used to analyze the prokaryotic expression characteristics for the gene. The analysis results of sequence indicated that the full-length cDNA of BcSGT1 gene was 1 418 bp, and the open reading frame was 1 074 bp in length, encoding a total of 358 amino acids.The relative molecular weight was 39.77 kD, and the protein theoretical isoelectric point was 5.05(GenBank No. AB495003). The evolutionary analysis of amino acid homologous system showed that the BcSGT1 gene of the non-heading Chinese cabbage had similar evolutionary relationship with the same family plant, and the highest homology(97%) was found with the chromosome 3 gene of Brassica rapa(Bra000741). The qRT-PCR analysis showed that under the infection of P. parasitica, the expression level of BcSGT1 gene in the resistant variety 'Suzhouqing' peaked at the 24 h, while the expression level in the susceptible variety 'Aijiaohuang'reached the peak at the 48 h. The peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.1 times higher than that in the susceptible variety 'Aijiaohuang'(P<0.01). With the infection of A.brassicicola, the expression level of BcSGT1 in 'Suzhouqing' reached its peak at 12 h, while the expression level in the susceptible variety 'Aijiaohuang' peaked at 24 h, and the peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.0 times higher than the peak expression in the susceptible variety'Aijiaohuang'(P<0.01). The expression level of BcSGT1 in 'Suzhouqing' is significantly higher than the expression level in 'Aijiaohuang' after 24 and 48 hours of infection by P. parasitica, and 12 h, 24 h, 48 h infection by A. brassicicola(P<0.01). The prokaryotic expression vector was induced by isopropyl β-Dthiogalactoside(IPTG) to express a fusion protein with a relative molecular mass of about 39 kD. The BcSGT1 gene of non-heading Chinese cabbage played an important role in the infection of P. parasitica and A.brassicicola, and BcSGT1 successfully achieved fusion expression in Escherichia coli, which provided the conditions for further protein level research and transgenic function research. It will also provide an important theoretical value for the selection of high-yield, high-quality and disease-related non-heading Chinese cabbage varieties.
引文
陈晓峰.2008.不结球白菜抗真菌病基因的克隆与表达分析[D].博士学位论文,南京农业大学,导师:侯喜林.pp.21-27.(Chen X F.2008.Molecular characterization of antifungal genes in Brassica campestris ssp.chinensis[D].Thesis for Ph.D.,Nanjing Agricultural University,Supervisor:Hou X L,pp.21-27.)
蒋明,张志仙,潘小翠,等.2015.青花菜抗病防卫基因BoS-GT1的克隆、序列分析与诱导表达[J].浙江大学学报(理学版),42(04):453-458.(Jiang M,Zhang Z X,Pan XC,et al.2015.Isolation,sequence analysis and induced expression of a broccoli disease defense gene BoSGT1[J].Journal of Zhejiang University(Science Edition),42(4):453-458.)
黄菲艺,唐君,侯喜林,等.2015.不结球白菜响应ABA和低温基因WRKY18的克隆及表达分析[J].南京农业大学学报,38(02):189-196.(Huang F Y,Tang J,Hou X L,et al.2015.Cloning and expression analysis of WRKY18gene in non-heading Chinese cabbage under ABA and low temperature treatments[J].Journal of Nanjing Agricultural University,38(02):189-196.)
屠煦童,张仕杰,吕东,等.2013.MhRAR1和MhSGT1基因转化苹果提高轮纹病菌诱导的抗氧化酶活性[J].园艺学报,40(12):2354-2364.(Tu X T,Zhang S J,LüD,et al.2013.Transformation of Malus×domestica with MhRAR1and MhSGT1 genes from Malus hupehensis for resistance of Botryosphaeria berengeriana[J].Acta Horticulturae Sinica,40(12):2354-2364.)
王超,张晓烜,王宁宁,等.2012.甘蓝黑斑病病原菌鉴定[C]//.中国园艺学会十字花科蔬菜分会,中国园艺学会十字花科蔬菜分会第十届学术研讨会论文集.中国园艺学会十字花科蔬菜分会,中国,pp.5.(Wang C,Zhang X X,Wang N N,et al.2012.Identiffication of blackspot disease pathogen in cabbage[C]//.Chinese Horticultural Society Cruciferous Vegetable Branch,Proceedings of the 10th Symposium of the Cruciferous Vegetable Branch of the Chinese Horticultural Society.Chinese Horticultural Society Cruciferous Vegetable Branch,CHN,pp.5.)
王凯,张增艳,黄璜,等.2007.小麦SGT1基因的克隆与表达特性分析[J].麦类作物学报,27(06):952-956.(Wang K,Zhang Z Y,Huang H,et al.2007.Cloning and expression analysis of SGT1 gene in Triticum aestivum[J].Journal of Triticeae Crops,27(06):952-956.)
王凯,张增艳.2008.SGT1在植物抗病反应中的功能研究进展[J].植物遗传资源学报,9(01):115-118.(Wang K,Zhang Z Y.2008.Study progress of SGT1 function on plant resistant response[J].Journal of Plant Genetic Resources,9(01):115-118.)
肖长坤,李勇,李健强.2003.十字花科蔬菜种传黑斑病研究进展[J].中国农业大学学报,8(5):61-68.(Xiao C K,Li Y,Li J Q.2003.Research in seed-borne black spot disease in cruciferous vegetables[J].Journal of China Agricultural University,8(5):61-68.)
肖栋,韦艳萍,李英,等.2018.不结球白菜病程相关蛋白基因BcPR5的克隆及表达分析[J].南京农业大学学报,41(4):640-646.(Xiao D,Wei Y P,Li Y,et al.2018.Cloning and expression analysis of pathogenesis-related protein gene BcPR5 in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,8(5):61-68.)
Azevedo C,Betsuyaku S,Peart J,et al.2006.Role of SGT1in resistance protein accumulation in plant immunity[J].Embo Journal,25(9):2007-2016.
Azevedo C,Sadanondom A,Kitagawa K,et al.2002.The RAR1 interactor SGT1,an essential component of Rgene-triggered disease resistance[J].Science,295:2073-2076.
Belkhadir Y,Subramaniam R,Dangl J L.2004.Plant disease resistance protein signaling:NBS-LRR proteins and their partners[J].Current Opinion in Plant Biology,7(4):391-399.
Das A K,Cohen P W,Barford D.1998.The structure of the tetratricopeptide repeats of protein phosphatase 5:Implications for TPR-mediated protein-protein interactions[J].The Embo Journal,17(5):1192-1199.
Kadota Y,Shirasu K,Guerois.2010.NLR sensors meet at the SGT1-HSP90 crossroad[J].Trends in Biochemical Sciences,35(4):199-207.
Kud J,Zhao Z,Du X,et al.2013.SGT1 interacts with the Prf resistance protein and is required for Prf accumulation and Prf-mediated defense signaling[J].Biochemical&Biophysical Research Communications,431(3):501-505.
Mchale L,Tan X,Koehl P,et al.2006.Plant NBS-LRR proteins:Adaptable guards[J].Genome Biology,7(4):1-11.
Moffett P,Farnham G,Peart J,et al.2002.Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death[J].Embo Journal,21(17):4511-4519.
Peart J R,Lu R,Sadanandom A,et al.2002.Ubiquitin ligaseassociated protein SGT1 is required for host and nonhost disease resistance in plants[J].Proceedings of the National Academy of Sciences of the USA,99(16):10865-10869.
Seo Y S,Lee S K,Song M Y,et al.2008.The HSP90-SGT1-RAR1 molecular chaperone complex:A core modulator in plant immunity[J].Journal of Plant Biology,51(1):1-10.
Tashakkori M M,Tebianian M,Tabatabaei M,et al.2016.Cloning,expression,and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system[J].International Journal of Mycobacteriology,(5):S249.
Takahashi A,Casais C,Ichimura K,et al.2003.HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis[J].Proceedings of the National Academy of Sciences of the USA,100(20):11777-11782.
Tamura K,Peterson D,Peterson N.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology&Evolution,28(10):2731-2739.
Uppalapati S R,Ishiga Y,Ryu C M,et al.2011.SGT1 contributes to coronatine signaling and Pseudomonas syringae pv.tomato disease symptom development in tomato and Arabidopsis[J].New Phytologist,189(1):83-93.
Xiao D,Liu S T,Wei Y P,et al.2016.cDNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica[J].Horticulture Research,3:16034.
Xiao D,Wang H,Basnet R K,et al.2014.Genetic dissection of leaf development in Brassica rapa using a genetical genomics approach[J].Plant Physiology,164(3):1309-1325.
蒋明,张志仙,潘小翠,等.2015.青花菜抗病防卫基因BoS-GT1的克隆、序列分析与诱导表达[J].浙江大学学报(理学版),42(04):453-458.(Jiang M,Zhang Z X,Pan XC,et al.2015.Isolation,sequence analysis and induced expression of a broccoli disease defense gene BoSGT1[J].Journal of Zhejiang University(Science Edition),42(4):453-458.)
黄菲艺,唐君,侯喜林,等.2015.不结球白菜响应ABA和低温基因WRKY18的克隆及表达分析[J].南京农业大学学报,38(02):189-196.(Huang F Y,Tang J,Hou X L,et al.2015.Cloning and expression analysis of WRKY18gene in non-heading Chinese cabbage under ABA and low temperature treatments[J].Journal of Nanjing Agricultural University,38(02):189-196.)
屠煦童,张仕杰,吕东,等.2013.MhRAR1和MhSGT1基因转化苹果提高轮纹病菌诱导的抗氧化酶活性[J].园艺学报,40(12):2354-2364.(Tu X T,Zhang S J,LüD,et al.2013.Transformation of Malus×domestica with MhRAR1and MhSGT1 genes from Malus hupehensis for resistance of Botryosphaeria berengeriana[J].Acta Horticulturae Sinica,40(12):2354-2364.)
王超,张晓烜,王宁宁,等.2012.甘蓝黑斑病病原菌鉴定[C]//.中国园艺学会十字花科蔬菜分会,中国园艺学会十字花科蔬菜分会第十届学术研讨会论文集.中国园艺学会十字花科蔬菜分会,中国,pp.5.(Wang C,Zhang X X,Wang N N,et al.2012.Identiffication of blackspot disease pathogen in cabbage[C]//.Chinese Horticultural Society Cruciferous Vegetable Branch,Proceedings of the 10th Symposium of the Cruciferous Vegetable Branch of the Chinese Horticultural Society.Chinese Horticultural Society Cruciferous Vegetable Branch,CHN,pp.5.)
王凯,张增艳,黄璜,等.2007.小麦SGT1基因的克隆与表达特性分析[J].麦类作物学报,27(06):952-956.(Wang K,Zhang Z Y,Huang H,et al.2007.Cloning and expression analysis of SGT1 gene in Triticum aestivum[J].Journal of Triticeae Crops,27(06):952-956.)
王凯,张增艳.2008.SGT1在植物抗病反应中的功能研究进展[J].植物遗传资源学报,9(01):115-118.(Wang K,Zhang Z Y.2008.Study progress of SGT1 function on plant resistant response[J].Journal of Plant Genetic Resources,9(01):115-118.)
肖长坤,李勇,李健强.2003.十字花科蔬菜种传黑斑病研究进展[J].中国农业大学学报,8(5):61-68.(Xiao C K,Li Y,Li J Q.2003.Research in seed-borne black spot disease in cruciferous vegetables[J].Journal of China Agricultural University,8(5):61-68.)
肖栋,韦艳萍,李英,等.2018.不结球白菜病程相关蛋白基因BcPR5的克隆及表达分析[J].南京农业大学学报,41(4):640-646.(Xiao D,Wei Y P,Li Y,et al.2018.Cloning and expression analysis of pathogenesis-related protein gene BcPR5 in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,8(5):61-68.)
Azevedo C,Betsuyaku S,Peart J,et al.2006.Role of SGT1in resistance protein accumulation in plant immunity[J].Embo Journal,25(9):2007-2016.
Azevedo C,Sadanondom A,Kitagawa K,et al.2002.The RAR1 interactor SGT1,an essential component of Rgene-triggered disease resistance[J].Science,295:2073-2076.
Belkhadir Y,Subramaniam R,Dangl J L.2004.Plant disease resistance protein signaling:NBS-LRR proteins and their partners[J].Current Opinion in Plant Biology,7(4):391-399.
Das A K,Cohen P W,Barford D.1998.The structure of the tetratricopeptide repeats of protein phosphatase 5:Implications for TPR-mediated protein-protein interactions[J].The Embo Journal,17(5):1192-1199.
Kadota Y,Shirasu K,Guerois.2010.NLR sensors meet at the SGT1-HSP90 crossroad[J].Trends in Biochemical Sciences,35(4):199-207.
Kud J,Zhao Z,Du X,et al.2013.SGT1 interacts with the Prf resistance protein and is required for Prf accumulation and Prf-mediated defense signaling[J].Biochemical&Biophysical Research Communications,431(3):501-505.
Mchale L,Tan X,Koehl P,et al.2006.Plant NBS-LRR proteins:Adaptable guards[J].Genome Biology,7(4):1-11.
Moffett P,Farnham G,Peart J,et al.2002.Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death[J].Embo Journal,21(17):4511-4519.
Peart J R,Lu R,Sadanandom A,et al.2002.Ubiquitin ligaseassociated protein SGT1 is required for host and nonhost disease resistance in plants[J].Proceedings of the National Academy of Sciences of the USA,99(16):10865-10869.
Seo Y S,Lee S K,Song M Y,et al.2008.The HSP90-SGT1-RAR1 molecular chaperone complex:A core modulator in plant immunity[J].Journal of Plant Biology,51(1):1-10.
Tashakkori M M,Tebianian M,Tabatabaei M,et al.2016.Cloning,expression,and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system[J].International Journal of Mycobacteriology,(5):S249.
Takahashi A,Casais C,Ichimura K,et al.2003.HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis[J].Proceedings of the National Academy of Sciences of the USA,100(20):11777-11782.
Tamura K,Peterson D,Peterson N.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology&Evolution,28(10):2731-2739.
Uppalapati S R,Ishiga Y,Ryu C M,et al.2011.SGT1 contributes to coronatine signaling and Pseudomonas syringae pv.tomato disease symptom development in tomato and Arabidopsis[J].New Phytologist,189(1):83-93.
Xiao D,Liu S T,Wei Y P,et al.2016.cDNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica[J].Horticulture Research,3:16034.
Xiao D,Wang H,Basnet R K,et al.2014.Genetic dissection of leaf development in Brassica rapa using a genetical genomics approach[J].Plant Physiology,164(3):1309-1325.
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