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1、生物工程學(xué)報(bào)ChinJBiotech2009,June25;25(6):819-825journals.im.ac.cnChineseJournalofBiotechnologyISSN1000-3061cjb@im.ac.cn?2009InstituteofMicrobiology,CAS&CSM,Allrightsreserved農(nóng)業(yè)生物技術(shù)枯草芽孢桿菌CAS15嗜鐵素基因dhbC的克隆、表達(dá)及功能鑒定1,22,32,321余賢美,林超,鄭服叢,賀春萍,張修國(guó)1山東農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院,泰安2
2、710182中國(guó)熱帶農(nóng)業(yè)科學(xué)院環(huán)境與植物保護(hù)研究所,儋州5717373海南大學(xué)環(huán)境與植物保護(hù)學(xué)院,儋州571737摘要:通過(guò)PCR技術(shù)擴(kuò)增得到dhbC基因,對(duì)其進(jìn)行序列分析發(fā)現(xiàn),dhbC基因片段長(zhǎng)為1197bp,預(yù)期編碼398個(gè)氨基酸,蛋白分子量大小為43.8kD。將目的片段連接到表達(dá)載體pET-30a(+),轉(zhuǎn)化大腸桿菌EscherichiacoliBL21(DE3)o獲得重組菌株BL21(DE3)/pET-30a-dhbC,以IPTG在30C誘導(dǎo)4h實(shí)現(xiàn)高效表達(dá),獲得一個(gè)分子量為48.8kD的
3、融合蛋白。重組蛋白可溶性分析結(jié)果表明:融合蛋白主要為可溶性蛋白。Westernblotting分析結(jié)果表明:重組蛋白可與兔抗His-tag多克隆抗體發(fā)生特異性反應(yīng),在48.8kD處有特異條帶,與預(yù)期結(jié)果一致,證明重組質(zhì)粒中含有dhbC基因。通過(guò)同源重組的策略將dhbC基因敲除后重新導(dǎo)入,驗(yàn)證了dhbC基因與嗜鐵素的生物合成密切相關(guān)。關(guān)鍵詞:枯草芽孢桿菌,dhbC基因,基因克隆,序列分析,原核表達(dá),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
