柑桔黄龙病菌亚洲种菌毛形成蛋白基因序列分析、原核表达及抗血清制备
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摘要
柑桔黄龙病菌菌毛形成蛋白(pilus formation protein, 408)是一种丰度较高的分泌蛋白。以感染柑桔黄龙病(HLB)的海南琼海市绿橙叶片为材料提取总DNA,利用408基因的特异引物进行PCR扩增,获得该基因的目的片段。序列分析结果表明海南琼海黄龙病菌408基因与柑桔黄龙病菌亚洲种psy62株系 (GenBank登录号:CP001677.5)408基因序列一致。功能预测结果表明其含有一个与菌毛形成相关的N端结构域,以及C端含有两个高度保守的基序(Motif 1和Motif 2)。通过Eco R Ⅴ和Xho Ⅰ双酶切构建重组载体p ET32a-408并将其转化BL21(DE3)大肠杆菌。重组菌经终浓度为1 mmoL/L IPTG诱导,目的蛋白主要以包涵体形式表达。目的蛋白经Ni2+-NTA层析柱纯化,并以此为抗原,腹腔免疫小白鼠,获得效价在1∶500～1∶10000的多克隆抗血清;Western blot进一步分析表明,408蛋白多克隆抗血清特异性强。研究结果为柑桔黄龙病菌408蛋白功能研究和柑桔黄龙病菌的蛋白检测产品开发提供重要科学依据。
Pilus formation protein(408) is a secreted protein involved in the assembly of bacterial pili with high-level expression. The total DNA was extracted from the HLB-infected green orange leaves of Qionghai City, Hainan Province, China. The target fragment of 408 gene was obtained by PCR amplification with specific primersand sequence analysis showed that the 408 gene is identical with the homologue gene of the Candidatus Liberibacter asiaticus psy62 isolate(GenBank accession number: CP001677.5). Functional prediction indicated that the protein contains a domain associated with Pilus formation protein at N terminal, while its C terminal contains two conserved motifs, Motif 1 and Motif 2. The recombinant vector pET32 a-408 was constructed by using Eco R Ⅴ and Xho Ⅰ restriction enzyme sites. The pET32 a-408 vector was transformed into E. coli BL21(DE3) and recombinant strain was induced by a final concentration of 1 mmoL/L IPTG. The 408 fusion protein was mainly expressed in the form of inclusion bodies. The 408 fusion protein was purified by Ni2+-NTA column and used to immunize mice by intraperitoneal injection. The 408 polyclonal antiserum was obtained and the titer was 1:500 ～ 1:10000. Western blotting further analysis showed that 408 polyclonal antiserum was specific to 408 protein. This study provides a research basis for the function study of 408 protein and the development of protein detection products for HLB.
Pilus formation protein(408) is a secreted protein involved in the assembly of bacterial pili with high-level expression. The total DNA was extracted from the HLB-infected green orange leaves of Qionghai City, Hainan Province, China. The target fragment of 408 gene was obtained by PCR amplification with specific primersand sequence analysis showed that the 408 gene is identical with the homologue gene of the Candidatus Liberibacter asiaticus psy62 isolate(GenBank accession number: CP001677.5). Functional prediction indicated that the protein contains a domain associated with Pilus formation protein at N terminal, while its C terminal contains two conserved motifs, Motif 1 and Motif 2. The recombinant vector pET32 a-408 was constructed by using Eco R Ⅴ and Xho Ⅰ restriction enzyme sites. The pET32 a-408 vector was transformed into E. coli BL21(DE3) and recombinant strain was induced by a final concentration of 1 mmoL/L IPTG. The 408 fusion protein was mainly expressed in the form of inclusion bodies. The 408 fusion protein was purified by Ni2+-NTA column and used to immunize mice by intraperitoneal injection. The 408 polyclonal antiserum was obtained and the titer was 1:500 ～ 1:10000. Western blotting further analysis showed that 408 polyclonal antiserum was specific to 408 protein. This study provides a research basis for the function study of 408 protein and the development of protein detection products for HLB.
引文
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[14]侯卫真.柑桔黄龙病Serralysin蛋白的表达纯化及两种重要病原的分子检测[D].武汉:华中农业大学,2014.
[15]Duan Y, Zhou L, Hall D G, et al. Complete genomesequenceofcitrushuanglongbing bacterium,‘Candidatus Liberibacter asiaticus’obtained through metagenomics[J]. Mol Plant Microbe Interact, 2009, 22(8):1011-1020.
[16]余乃通,冯团诚,王健华,等.香蕉束顶病毒海口分离物Rep基因的克隆、原核表达、抗血清制备及检测[J].植物保护, 2011,37(4):38-43.
[17]Li W, Hartung J S, Levy L. Quantitative realtime PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing[J]. J Microbiol Methods,2006, 66(1):104-115.
[18]OkudaM,MatsumotoM,TanakaY,etal.CharacterizationofthetufB-secE-nusGrplKAJL-rpoB gene cluster of the citrus greening organismanddetectionbyloop-mediated isothermal amplification[J]. Plant Disease,2007, 89(7):705-711.
[19]Arratia-Castro A A, Santos-Cervantes M E,Arce-LealáP, et al. Detection and quantification of‘CandidatusPhytoplasmaasteris?and‘Candidatus Liberibacter asiaticus?at early and late stages of Huanglongbing disease development[J]. Canadian journal of plant pathology, 2016,38(4):411-421.
[20]Qian W, Lu Y, Meng Y, et al. Field Detection of Citrus Huanglongbing Associated with ‘Candidatus Liberibacter Asiaticus’by Recombinese Polymerase Amplification within 15 min[J]. Journal of Agricultural and Food Chemistry, 2018, 66(22):5473-5480.
[21]高玉霞.柑桔黄龙病PCR检测引物比较及其外膜蛋白抗体的研制[D].赣州:赣南师范学院,2015.
[22]Yuan Q, Jordan R, Brlansky R H, et al. Development of single chain variable fragment (scFv) antibodies against surface proteins of‘Ca. Liberibacter asiaticus’[J]. Journal of Microbiological Methods,2016, 122:1-7.
[23]Liu H, Atta S, Hartung J S. Characterization and purification of proteins suitable for the production of antibodies against‘Ca. Liberibacter asiaticus’[J]. Protein Expression&Purification, 2017,139(5):36-42.
[24]董俊.湖南省柑桔黄龙病调查及病菌遗传多样性分析[D].长沙:湖南农业大学,2015.
[2]Cevallos-Cevallos J M, Futch D B, Shilts T, et al.GC-MS metabolomic differentiation of selected citrus varieties with different sensitivity to citrus huanglongbing[J]. Plant Physiol Biochem, 2012,53(4):69-76.
[3]DaGJ,DouhanGW,HalbertSE,etal.Huanglongbing:Anoverviewofacomplex pathosystem ravaging the world's citrus[J].Journal of Integrative Plant Biology, 2016, 58(4):373-387.
[4]谭锦.中国柑桔黄龙病病原菌原噬菌体遗传多样性研究[D].重庆:西南大学,2013.
[5]程保平,鹿连明,彭埃天,等.柑桔多个品种和多个部位中黄龙病菌的检测与调查[J].广东农业科学,2014,41(11):69-72.
[6]BovéJ M. Huanglongbing:a destructive, newlyemerging, century-old disease of citrus[J].Journal of Plant Pathology,2006,88(1):7-37.
[7]Gottwald T R, Gra?a J V D, Bassanezi R B. Citrus Huanglongbing:the pathogen and its impact[J].Plant Health Progress, 2007:906-910.
[8]WuX,MengC,WangG,etal.Rapidand quantitative detection of citrus huanglongbing bacterium‘Candidatus, Liberibacter asiaticus’by real-time fluorescent loop-mediated isothermal amplification assay in China[J]. Physiological&Molecular Plant Pathology, 2016, 94:1-7.
[9]Li B, Yang Y, Liu Z, et al.柑桔黄龙病菌(Candidatus Liberibacter asiaticus)分泌蛋白基因DNA含量及其RNA表达量关系分析//中国植物病理学会2018年学术年会论文集[C]. 2018.
[10]Costa T R, Felisberto-Rodrigues C, Meir A, et al. Secretion systems in Gram-negative bacteria:structural and mechanistic insights[J]. Nature Reviews Microbiology, 2015, 13(6):343-359.
[11]Abby S S, Cury J, Guglielmini J, et al. Identification of protein secretion systems in bacterial genomes[J]. Sci Rep, 2016, 6:1-21.
[12]Guglielmini J, Néron B, Abby S S, et al. Key components of the eight classes of typeⅣsecretion systems involved in bacterial conjugation or protein secretion[J]. Nucleic Acids Research,2014, 42(9):5715-5727.
[13]Clark K, Franco J Y, Schwizer S, et al. An effector from the Huanglongbing-associated pathogen targets citrus proteases[J]. Nature Communications, 2018, 9(1):1718.
[14]侯卫真.柑桔黄龙病Serralysin蛋白的表达纯化及两种重要病原的分子检测[D].武汉:华中农业大学,2014.
[15]Duan Y, Zhou L, Hall D G, et al. Complete genomesequenceofcitrushuanglongbing bacterium,‘Candidatus Liberibacter asiaticus’obtained through metagenomics[J]. Mol Plant Microbe Interact, 2009, 22(8):1011-1020.
[16]余乃通,冯团诚,王健华,等.香蕉束顶病毒海口分离物Rep基因的克隆、原核表达、抗血清制备及检测[J].植物保护, 2011,37(4):38-43.
[17]Li W, Hartung J S, Levy L. Quantitative realtime PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing[J]. J Microbiol Methods,2006, 66(1):104-115.
[18]OkudaM,MatsumotoM,TanakaY,etal.CharacterizationofthetufB-secE-nusGrplKAJL-rpoB gene cluster of the citrus greening organismanddetectionbyloop-mediated isothermal amplification[J]. Plant Disease,2007, 89(7):705-711.
[19]Arratia-Castro A A, Santos-Cervantes M E,Arce-LealáP, et al. Detection and quantification of‘CandidatusPhytoplasmaasteris?and‘Candidatus Liberibacter asiaticus?at early and late stages of Huanglongbing disease development[J]. Canadian journal of plant pathology, 2016,38(4):411-421.
[20]Qian W, Lu Y, Meng Y, et al. Field Detection of Citrus Huanglongbing Associated with ‘Candidatus Liberibacter Asiaticus’by Recombinese Polymerase Amplification within 15 min[J]. Journal of Agricultural and Food Chemistry, 2018, 66(22):5473-5480.
[21]高玉霞.柑桔黄龙病PCR检测引物比较及其外膜蛋白抗体的研制[D].赣州:赣南师范学院,2015.
[22]Yuan Q, Jordan R, Brlansky R H, et al. Development of single chain variable fragment (scFv) antibodies against surface proteins of‘Ca. Liberibacter asiaticus’[J]. Journal of Microbiological Methods,2016, 122:1-7.
[23]Liu H, Atta S, Hartung J S. Characterization and purification of proteins suitable for the production of antibodies against‘Ca. Liberibacter asiaticus’[J]. Protein Expression&Purification, 2017,139(5):36-42.
[24]董俊.湖南省柑桔黄龙病调查及病菌遗传多样性分析[D].长沙:湖南农业大学,2015.