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1、生物工程学报ChinJBiotech2009,June25;25(6):819-825journals.im.ac.cnChineseJournalofBiotechnologyISSN1000-3061cjb@im.ac.cn©2009InstituteofMicrobiology,CAS&CSM,Allrightsreserved农业生物技术枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定1,22,32,321余贤美,林超,郑服丛,贺春萍,张修国1山东农业大学植物保护学院,泰安2
2、710182中国热带农业科学院环境与植物保护研究所,儋州5717373海南大学环境与植物保护学院,儋州571737摘要:通过PCR技术扩增得到dhbC基因,对其进行序列分析发现,dhbC基因片段长为1197bp,预期编码398个氨基酸,蛋白分子量大小为43.8kD。将目的片段连接到表达载体pET-30a(+),转化大肠杆菌EscherichiacoliBL21(DE3)o获得重组菌株BL21(DE3)/pET-30a-dhbC,以IPTG在30C诱导4h实现高效表达,获得一个分子量为48.8kD的
3、融合蛋白。重组蛋白可溶性分析结果表明:融合蛋白主要为可溶性蛋白。Westernblotting分析结果表明:重组蛋白可与兔抗His-tag多克隆抗体发生特异性反应,在48.8kD处有特异条带,与预期结果一致,证明重组质粒中含有dhbC基因。通过同源重组的策略将dhbC基因敲除后重新导入,验证了dhbC基因与嗜铁素的生物合成密切相关。关键词:枯草芽孢杆菌,dhbC基因,基因克隆,序列分析,原核表达,基因敲除Cloning,expressionandfunctionalanalysisofthedhb
4、CgenefromthesiderophoreproducingbacteriumBacillussubtilisCAS151,22,32,321XianmeiYu,ChaoLin,FucongZheng,ChunpingHe,andXiuguoZhang1PlantProtectionCollege,ShandongAgriculturalUniversity,Tai’an271018,China2EnvironmentandPlantProtectionInstitute,ChineseAca
5、demyofTropicalAgriculturalSciences,Danzhou571737,China3EnvironmentandPlantProtectionCollege,HainanUniversity,Danzhou571737,ChinaAbstract:WeamplifieddhbCgenefromthesiderophoreproducingbacteriumCAS15byPCR.AfterligatedthePCRproducttopMD18-Tvectorandthens
6、equenced,weobtaineda1197bpfragment.TheblastresultshowedthatthenucleotideacidsofdhbCgene(AccessionNo.FJ194456)ofCAS15shared99.7%identitywiththatofdhbCgeneofBacillussubtilis(GenBankAccessionNo.Z99120),andwaspredictedtoencodea43.8kDpolypeptidewith398amin
7、oacidresidues.WeclonedthedhbCgeneintoexpressionvectorpET-30a(+)andthentransformedintoEscherichiacoliBL21(DE3)viacalciumchloridetransformationmethod,andobtainedReceived:September15,2008;Accepted:March16,2009Supportedby:CentralBasicR&DSpecialFundforPubl
8、icWelfareInstitutesofChina(Nos.2008hzs1J013,2008hzs1J014),NationalSupportProjectforScienceandTechnology(No.2007BAD48B04),NationalSpecialFundforPublicWelfareIndustry(No.nyhyzx07-033-2-3).Correspondingauthor:XiuguoZhang.Tel:+86-538-8249095;E-mai
