柑橘黄龙病病原CLIBASIA＿03230基因的克隆及其编码蛋白的原核表达
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摘要
【目的】柑橘黄龙病(Citrus Huanglongbing,HLB)是由韧皮部杆菌(Candidatus Liberibacter spp.)引发的重要柑橘病害。本试验利用生物信息学方法分析韧皮部杆菌全基因组,推测CLIBASIA_03230基因(命名为Clsp11)编码分泌蛋白,克隆该基因并构建其原核表达载体,表达、纯化Clsp11蛋白,以用于制备特异抗体。【方法】利用CTAB法从感染柑橘黄龙病病原的长春花中提取总DNA,以此为模板经PCR扩增Clsp11,并克隆至pMD18-T载体,挑取阳性克隆测序验证;利用无缝克隆技术将Clsp11克隆至原核表达载体pET30a,通过PCR和酶切鉴定筛选阳性克隆,命名为pET30a-Clsp11;转化大肠杆菌BL21,经IPTG诱导,表达N-端融合6×His标签的Clsp11重组蛋白,SDS-PAGE电泳检测,分析目的蛋白的表达水平,并利用镍柱进行纯化。【结果】Clsp11蛋白成功表达,IPTG诱导5 h表达量显著提高,主要以包涵体形式存在于宿主菌中。【结论】本研究克隆了柑橘黄龙病病原Clsp11基因,构建了原核表达载体pET30a-Clsp11,并成功表达、纯化了Clsp11重组蛋白,为进一步制备特异性抗体和研究Clsp11生物学功能奠定了基础。
[Purpose]Pathogenic bacteria secreted proteins play an important role in the process of pathogen infection. Analysis of the biological functions of secretory proteins will help to clarify the molecular mechanism of pathogen-plant interaction. In this study, the entire genome of Candidatus Liberibacter asiaticus was subjected to bioinformatics analysis, and CLIBASIA_03230 gene(named as Clsp11) was predicted to encode a secreted protein. Therefore, the Clsp11 was cloned and inserted into the prokaryotic expression vector, and the Clsp11 protein was expressed and purified for preparation of the specific antibodies in the next step.[Method]Total DNA was extracted from Ca. L. asiaticus-infected periwinkle, and was used as template to amplify Clsp11. The resulting PCR products were cloned into pMD18-T vector and sequenced. With the In-Fusion cloning technique, the Clsp11 was inserted into the pET30 a, resulting in pET30 a-Clsp11. The pET30 a-Clsp11 was subsequently transformed into Escherichia coli BL21 and induced by IPTG to express the recombinant protein Clsp11, the N-terminus of which was fused with 6×His tag. After analysis with SDS-PAGE, the Clsp11 recombinant protein was purified by nickel column.[Result]The Clsp11 protein was successfully expressed, and reached high expression level when IPTG induction for 5 hours. Additionally, a large portion of the recombinant proteins present as inclusion bodies.[Conclusion]In this study, the Clsp11 of Ca. L. asiaticus was cloned, the prokaryotic expression vector pET30 a-Clsp11 was constructed, and the recombinant protein Clsp11 was expressed and purified, thus laying the foundation to prepare specific antibodies as well as investigate the biological function(s) of Clsp11.
[Purpose]Pathogenic bacteria secreted proteins play an important role in the process of pathogen infection. Analysis of the biological functions of secretory proteins will help to clarify the molecular mechanism of pathogen-plant interaction. In this study, the entire genome of Candidatus Liberibacter asiaticus was subjected to bioinformatics analysis, and CLIBASIA_03230 gene(named as Clsp11) was predicted to encode a secreted protein. Therefore, the Clsp11 was cloned and inserted into the prokaryotic expression vector, and the Clsp11 protein was expressed and purified for preparation of the specific antibodies in the next step.[Method]Total DNA was extracted from Ca. L. asiaticus-infected periwinkle, and was used as template to amplify Clsp11. The resulting PCR products were cloned into pMD18-T vector and sequenced. With the In-Fusion cloning technique, the Clsp11 was inserted into the pET30 a, resulting in pET30 a-Clsp11. The pET30 a-Clsp11 was subsequently transformed into Escherichia coli BL21 and induced by IPTG to express the recombinant protein Clsp11, the N-terminus of which was fused with 6×His tag. After analysis with SDS-PAGE, the Clsp11 recombinant protein was purified by nickel column.[Result]The Clsp11 protein was successfully expressed, and reached high expression level when IPTG induction for 5 hours. Additionally, a large portion of the recombinant proteins present as inclusion bodies.[Conclusion]In this study, the Clsp11 of Ca. L. asiaticus was cloned, the prokaryotic expression vector pET30 a-Clsp11 was constructed, and the recombinant protein Clsp11 was expressed and purified, thus laying the foundation to prepare specific antibodies as well as investigate the biological function(s) of Clsp11.
引文
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[11]HIJAZ F M,MANTHEY J A,FOLIMONOVA S Y,et al.An HPLC-MS characterization of the changes in sweet orange leaf metabolite profile following infection by the bacterial pathogen Candidatus Liberibacter asiaticus[J].PLoS One,2013,8(11):e79485.DOI:10.1371/journal.pone.0079485.
[12]LI J Y,PANG Z Q,TRIVEDI P,et al.‘Candidatus Liberibacter asiaticus’encodes a functional salicylic acid(SA)hydroxylase that degrades SA to suppress plant defenses[J].Molecular Plant-Microbe Interactions,2017,30(8):620.DOI:10.1094/MPMI-12-16-0257-R.
[13]刘萍,陈苗苗,刘学荣,等.单克隆抗体研究进展[J].中国畜牧兽医,2012,39(1):67.DOI:10.3969/j.issn.1671-7236.2012.01.016.
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[15]PAGLIACCIA D,SHI J X,PANG Z Q,et al.A pathogen secreted protein as a detection marker for Citrus Huanglongbing[J].Frontiers in Microbiology,2017,8(2):2041.DOI:10.3389/fmicb.2017.02041.
[16]PETERSEN T N,BRUNAK S,VON HEIJNE G,et al.SignalP 4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785.DOI:10.1038/nmeth.1701.
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[18]MELHEM H,MIN X J,BUTLER G.The impact of SignalP 4.0 on the prediction of secreted proteins[J].Institute of Electrical and Electronics Engineers,2013:16.
[19]MAK M W,WANG W,KUNG S Y.Fusion of conditional random field and SignalP for protein cleavage site prediction[C].Asia-Pacific Signal and Information Processing Association,2009.
[20]祁春节,宋金田.2009年中国春茶产销形势分析[J].茶世界,2009,26(4):22.
[21]REINKING O A.Diseases of economic plants in southern China[J].Philippine Agricultural,1919,4(8):109.
[22]IREY M S,GAST T,GOTTWALD T R.Comparison of visual assessment and polymerase chain reaction assay testing to estimate the incidence of the Huanglongbing pathogem in commercial Florida citrus[J].State Hort,2006,119(1):89.
[23]DING F,DUAN Y P,PAUL C,et al.Localization and distribution of‘Candidatus Liberibacter asiaticus’in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody[J].PLoS One,2015,10(5):0123939.DOI:10.1371/journal.pone.0123939.
[24]GOTTWALD T R.Current epidemiological understanding of Citrus Huanglongbing[J].Annual Review of Phytopathology,2010,48(2):119.DOI:10.1146/annurevphyto-073009-114418.
[2]范国成,刘波,吴如健,等.中国柑橘黄龙病研究30年[J].福建农业学报,2009,24(2):183.DOI:10.3969/j.issn.1008-0384.2009.02.019.
[3]单杨.中国柑橘工业的现状、发展趋势与对策[J].中国食品学报,2008,8(1):1.DOI:10.3969/j.issn.1009-7848.2008.01.001.
[4]邓军蓉,祁春节.供应链环境下我国柑橘物流现状分析[J].农村经济,2009(2):50.DOI:10.3969/j.issn.1671-7252.2009.02.011.
[5]胡文召,周常勇.柑橘黄龙病病原研究进展[J].植物保护,2010,36(3):30.DOI:10.3969/j.issn.0529-1542.2010.03.007.
[6]DUAN Y P,ZHOU L J,HALL D G,et al.Complete genome sequence of citrus huanglongbing bacterium,‘Candidatus Liberibacter asiaticus’obtained through metagenomics[J].Molecular Plant-Microbe Interactions,2009,22(8):1011.DOI:10.1094/MPMI-22-8-1011.
[7]WULFF N A,ZHANG S J,SETUBAL J C,et al.The complete genome sequence of‘Candidatus Liberibacter americanus’,associated with Citrus huanglongbing[J].Molecular Plant-Microbe Interactions,2014,27(2):163.DOI:10.1094/MPMI-09-13-0292-R.
[8]PITINO M,ARMSTRONG C M,CANO L M,et al.Transient expression of Candidatus liberibacter asiaticus effector induces cell death in nicotiana benthamiana[J].Frontiers in Plant Science,2016,7:982.DOI:10.3389/fpls.2016.00982.
[9]PRASAD S,XU J,ZHANG Y Z,et al.SEC-Translocon dependent extracytoplasmic proteins of Candidatus liberibacter asiaticus[J].Frontiers in Microbiology,2016,7:1989.DOI:10.3389/fmicb.2016.01989.
[10]HAO G X,PITINO M,DING F,et al.Induction of innate immune responses by flagellin from the intracellular bacterium,‘Candidatus Liberibacter solanacearum’[J].BMC Plant Biology,2014,14(1):211.DOI:10.1186/s12870-014-0211-9.
[11]HIJAZ F M,MANTHEY J A,FOLIMONOVA S Y,et al.An HPLC-MS characterization of the changes in sweet orange leaf metabolite profile following infection by the bacterial pathogen Candidatus Liberibacter asiaticus[J].PLoS One,2013,8(11):e79485.DOI:10.1371/journal.pone.0079485.
[12]LI J Y,PANG Z Q,TRIVEDI P,et al.‘Candidatus Liberibacter asiaticus’encodes a functional salicylic acid(SA)hydroxylase that degrades SA to suppress plant defenses[J].Molecular Plant-Microbe Interactions,2017,30(8):620.DOI:10.1094/MPMI-12-16-0257-R.
[13]刘萍,陈苗苗,刘学荣,等.单克隆抗体研究进展[J].中国畜牧兽医,2012,39(1):67.DOI:10.3969/j.issn.1671-7236.2012.01.016.
[14]徐惠迪,蔡少华.单克隆抗体在植物病理学中的应用及其研究进展[J].生物技术通报,1988(3):1.
[15]PAGLIACCIA D,SHI J X,PANG Z Q,et al.A pathogen secreted protein as a detection marker for Citrus Huanglongbing[J].Frontiers in Microbiology,2017,8(2):2041.DOI:10.3389/fmicb.2017.02041.
[16]PETERSEN T N,BRUNAK S,VON HEIJNE G,et al.SignalP 4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785.DOI:10.1038/nmeth.1701.
[17]EMANUELSSON O,BRUNAK S,VON HEIJNE G,et al.Locating proteins in the cell using TargetP,SignalPand related tools[J].Nature Protocols,2007,2(4):953.DOI:10.1038/nprot.2007.131.
[18]MELHEM H,MIN X J,BUTLER G.The impact of SignalP 4.0 on the prediction of secreted proteins[J].Institute of Electrical and Electronics Engineers,2013:16.
[19]MAK M W,WANG W,KUNG S Y.Fusion of conditional random field and SignalP for protein cleavage site prediction[C].Asia-Pacific Signal and Information Processing Association,2009.
[20]祁春节,宋金田.2009年中国春茶产销形势分析[J].茶世界,2009,26(4):22.
[21]REINKING O A.Diseases of economic plants in southern China[J].Philippine Agricultural,1919,4(8):109.
[22]IREY M S,GAST T,GOTTWALD T R.Comparison of visual assessment and polymerase chain reaction assay testing to estimate the incidence of the Huanglongbing pathogem in commercial Florida citrus[J].State Hort,2006,119(1):89.
[23]DING F,DUAN Y P,PAUL C,et al.Localization and distribution of‘Candidatus Liberibacter asiaticus’in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody[J].PLoS One,2015,10(5):0123939.DOI:10.1371/journal.pone.0123939.
[24]GOTTWALD T R.Current epidemiological understanding of Citrus Huanglongbing[J].Annual Review of Phytopathology,2010,48(2):119.DOI:10.1146/annurevphyto-073009-114418.
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