植物乳桿菌質(zhì)粒表達(dá)系統(tǒng)的構(gòu)建
發(fā)布時(shí)間:2021-09-17 01:29
乳酸菌是人體腸道微生物組成成分,為普遍公認(rèn)的安全菌株,應(yīng)用于食品工業(yè)中,作為表達(dá)系統(tǒng)的宿主菌相比其他微生物有天然優(yōu)勢。本課題從傳統(tǒng)食品中分離到一株乳酸菌,經(jīng)過16S rRNA測序與序列比對,結(jié)合傳統(tǒng)的生理生化實(shí)驗(yàn),鑒定為植物乳桿菌的近緣種戊糖乳桿菌,命名為戊糖乳桿菌JS1(Lactobacillus pentosus JS1)。此菌株中含有一個(gè)大小為13kb的天然質(zhì)粒,使用引物步移法獲得質(zhì)粒的序列,拼接得到質(zhì)粒全長13205bp。通過與NCBI數(shù)據(jù)庫比對,確定質(zhì)粒為一個(gè)新的質(zhì)粒,命名為pJS1。其G+C含量為35%,有14個(gè)開放閱讀框。序列分析發(fā)現(xiàn),質(zhì)粒含有兩個(gè)復(fù)制子,分別命名為ori1與ori2。兩個(gè)復(fù)制子的序列相似達(dá)80%,ori1含有長度為22bp的4.8個(gè)重復(fù)正向重復(fù)序列;ori2含有4.5個(gè)重復(fù)正向重復(fù)序列。兩個(gè)重復(fù)序列僅兩個(gè)堿基差異。在復(fù)制子下游均緊接一個(gè)可能與復(fù)制子相關(guān)的repB。為確定最小復(fù)制子,將含有復(fù)制子序列的不同的片段克隆到大腸桿菌質(zhì)粒pUCM構(gòu)建大腸桿菌-乳酸菌穿梭載體。結(jié)果表明,兩個(gè)含復(fù)制子片段均可在胞內(nèi)復(fù)制,ori1的最小復(fù)制子包括完整的正向重復(fù)序列,完整的...
【文章來源】:華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁數(shù)】:79 頁
【學(xué)位級別】:碩士
【部分圖文】:
θ復(fù)制模型Fig.1Modelforθreplication注:大腸桿菌染色體(雙鏈環(huán)狀DNA)的復(fù)制
圖 2 滾環(huán)復(fù)制模型(del Solar Gloriadel 1998)Fig.2 Model for RC replicationThe plasmid-encoded Rep protein recognizes the dso on supercoiled DNAandintroduces a site-specific nick generating a free 3′-OH end. This end is elongated byhost proteins as the parental strand is being displaced. When the replication forkreaches the reconstituted dso, Rep protein catalyzes a strand transfer reaction,releasing an ssDNAintermediate and a dsDNAmolecule with a parental and anewly synthesized (dotted) strand. Lagging-strand synthesis on the ssDNAmolecule is initiated at the sso signal by the host RNApolymerase. This enzymewould synthesize a short primer RNA, and lagging-strand synthesis is performed byhost DNApolymerases. The end products are supercoiled plasmid DNAmolecules.鏈置換復(fù)制許多病毒是以鏈置換的方式復(fù)制,質(zhì)粒中也有少部分是以此方式復(fù)制,但乳酸菌中并沒有這樣的報(bào)導(dǎo)。
鏈置換復(fù)制模型(Brownetal2005)
本文編號:3397684
【文章來源】:華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁數(shù)】:79 頁
【學(xué)位級別】:碩士
【部分圖文】:
θ復(fù)制模型Fig.1Modelforθreplication注:大腸桿菌染色體(雙鏈環(huán)狀DNA)的復(fù)制
圖 2 滾環(huán)復(fù)制模型(del Solar Gloriadel 1998)Fig.2 Model for RC replicationThe plasmid-encoded Rep protein recognizes the dso on supercoiled DNAandintroduces a site-specific nick generating a free 3′-OH end. This end is elongated byhost proteins as the parental strand is being displaced. When the replication forkreaches the reconstituted dso, Rep protein catalyzes a strand transfer reaction,releasing an ssDNAintermediate and a dsDNAmolecule with a parental and anewly synthesized (dotted) strand. Lagging-strand synthesis on the ssDNAmolecule is initiated at the sso signal by the host RNApolymerase. This enzymewould synthesize a short primer RNA, and lagging-strand synthesis is performed byhost DNApolymerases. The end products are supercoiled plasmid DNAmolecules.鏈置換復(fù)制許多病毒是以鏈置換的方式復(fù)制,質(zhì)粒中也有少部分是以此方式復(fù)制,但乳酸菌中并沒有這樣的報(bào)導(dǎo)。
鏈置換復(fù)制模型(Brownetal2005)
本文編號:3397684
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