植物乳杆菌质粒表达系统的构建
发布时间：2021-09-17 01:29
乳酸菌是人体肠道微生物组成成分,为普遍公认的安全菌株,应用于食品工业中,作为表达系统的宿主菌相比其他微生物有天然优势。本课题从传统食品中分离到一株乳酸菌,经过16S rRNA测序与序列比对,结合传统的生理生化实验,鉴定为植物乳杆菌的近缘种戊糖乳杆菌,命名为戊糖乳杆菌JS1（Lactobacillus pentosus JS1）。此菌株中含有一个大小为13kb的天然质粒,使用引物步移法获得质粒的序列,拼接得到质粒全长13205bp。通过与NCBI数据库比对,确定质粒为一个新的质粒,命名为pJS1。其G+C含量为35%,有14个开放阅读框。序列分析发现,质粒含有两个复制子,分别命名为ori1与ori2。两个复制子的序列相似达80%,ori1含有长度为22bp的4.8个重复正向重复序列;ori2含有4.5个重复正向重复序列。两个重复序列仅两个碱基差异。在复制子下游均紧接一个可能与复制子相关的repB。为确定最小复制子,将含有复制子序列的不同的片段克隆到大肠杆菌质粒pUCM构建大肠杆菌-乳酸菌穿梭载体。结果表明,两个含复制子片段均可在胞内复制,ori1的最小复制子包括完整的正向重复序列,完整的...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：79 页
【学位级别】：硕士
【部分图文】：
θ复制模型Fig.1Modelforθreplication注：大肠杆菌染色体（双链环状DNA）的复制
图 2 滚环复制模型(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.链置换复制许多病毒是以链置换的方式复制，质粒中也有少部分是以此方式复制，但乳酸菌中并没有这样的报导。
链置换复制模型(Brownetal2005)
本文编号：3397684
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：79 页
【学位级别】：硕士
【部分图文】：
θ复制模型Fig.1Modelforθreplication注：大肠杆菌染色体（双链环状DNA）的复制
图 2 滚环复制模型(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.链置换复制许多病毒是以链置换的方式复制，质粒中也有少部分是以此方式复制，但乳酸菌中并没有这样的报导。
链置换复制模型(Brownetal2005)
本文编号：3397684
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