目的 将弓形虫SAG1基因截短片段 (t SAG1) ,分别用植物组成型E35S启动子和番茄果实特异性E8启动子驱动 ,构建t SAG1植物表达载体。方法 自本室构建的pET32a t SAG1载体中PCR扩增得到t SAG1基因 ,克隆进T载体为pMD T t SAG1,酶切鉴定...目的 将弓形虫SAG1基因截短片段 (t SAG1) ,分别用植物组成型E35S启动子和番茄果实特异性E8启动子驱动 ,构建t SAG1植物表达载体。方法 自本室构建的pET32a t SAG1载体中PCR扩增得到t SAG1基因 ,克隆进T载体为pMD T t SAG1,酶切鉴定后进行测序确认。用BamHⅠ单酶切连接方式将t SAG1分别亚克隆进 pB35MG、pB35E1MG、pBE2MG载体 ,分别构建中间载体 pB35 t SAG1、pB35E1 t SAG1、pBE2 t SAG1载体。其中 ,pB35 t SAG1以E35S驱动t SAG1,3′端携带NOS3′终止子序列 ,组成E35S/t SAG1/NOS3′操纵子结构单元。pB35E1 t SAG1含E35S和番茄果实特异性E8启动子核心序列E81.1双启动子驱动t SAG1,组成E35SE81.1/t SAG1/NOS3′操纵子结构单元。pBE2t SAG1以番茄果实特异性E8启动子全长E82 .2驱动t SAG1,组成E82 .2 /t SAG1/NOS3′操纵子结构单元。pB35 t SAG1、pB35E1 t SAG1、pBE2 t SAG1经酶切证实后 ,分别将其中E35S/t SAG1/NOS3′、E35S -E81.1/t SAG1/NOS3′、E82 .2 /t SAG1/NOS3′操纵子结构单元以HindⅢ单酶切连接方式亚克隆进pCAMBIA2 30 0质粒中 ,构建植物表达载体 pC35 t SAG1、pC35E1 t SAG1、pCE2 t SAG1,酶切鉴定。含三种启动子驱动t SAG1的植物表达载体转化根癌农杆菌LBA4 4 0 4感受态细胞后 ,展开更多
用Long accurate RT-PCR(LA-PCR)的方法一步法克隆了IBDV TL2004基因组A节段全长cDNA。序列测定结果表明：克隆的A节段全长3260个核苷酸,包括5′、3′的非编码区(NCRs)和2个重叠的开放阅读框(ORF1和ORF2),与来自GenBank IBDV血清Ⅰ型毒...用Long accurate RT-PCR(LA-PCR)的方法一步法克隆了IBDV TL2004基因组A节段全长cDNA。序列测定结果表明：克隆的A节段全长3260个核苷酸,包括5′、3′的非编码区(NCRs)和2个重叠的开放阅读框(ORF1和ORF2),与来自GenBank IBDV血清Ⅰ型毒株核苷酸序列的同源性高达95．2%～99．2%。二级结构分析表明,在5′-NCRs和3′-NCRs各存在一个大型的颈环发夹结构。ORF1编码1 012个氨基酸的VP2-VP3-VP4,在氨基酸水平上VP2、VP3、VP4与参比Ⅰ型毒株的同源性分别达96．7%～99．6%、94．2%～99．2%、96．7%～99．6%。PRF2编码145个氨基酸的VP5,与参比Ⅰ型毒株的同源性高达95．9%～99．3%。TL2004在第2个小亲水区内279位氨基酸由S替代了N、290位M替代了L,这2个突变可能是IBDV抗原漂变的原因。分子系统进化树分析表明,TL2004与欧洲Cu21株和中国JD1、Harbin株的关系较近,而与欧洲、香港、日本的超强毒株和美国的变异株相对较远。展开更多
Culturable thermophilic microorganisms were enriched from samples collected from Lau Basin hydrothermal vents in artificial seawater medium at 45 ℃ and pH 7.0. Microbial diversities of the enriched communities were d...Culturable thermophilic microorganisms were enriched from samples collected from Lau Basin hydrothermal vents in artificial seawater medium at 45 ℃ and pH 7.0. Microbial diversities of the enriched communities were defined by performing a restriction fragment length polymorphism （RFLP） analysis of 16S rRNA gene sequences with enzymes MspI and Hin6 I. A total of 14 phylotypes have been detected by the RFLP patterns identified for 16S rRNA clone libraries of the enrichment. Analysis of sequences showed that at least four bacterial divisions presented in the clones libraries. The phyla Proteobacteria and Firmicutes were the most dominant groups. The majority of the sequences included in this analysis affiliated with Gamma Proteobacteria （71%） and Bacillus （23%）. Scanning electron micrographs revealed that there were abundant rod and coceoidal forms encased in sulphur and sodium chloride precipitate. These results revealed that there were a diversity of moderate thermophilic bacterial populations thrived in Lau Basin hydrothermal vents that were previously not detected by either molecular retrieval or strain purification techniques.展开更多
文摘目的 将弓形虫SAG1基因截短片段 (t SAG1) ,分别用植物组成型E35S启动子和番茄果实特异性E8启动子驱动 ,构建t SAG1植物表达载体。方法 自本室构建的pET32a t SAG1载体中PCR扩增得到t SAG1基因 ,克隆进T载体为pMD T t SAG1,酶切鉴定后进行测序确认。用BamHⅠ单酶切连接方式将t SAG1分别亚克隆进 pB35MG、pB35E1MG、pBE2MG载体 ,分别构建中间载体 pB35 t SAG1、pB35E1 t SAG1、pBE2 t SAG1载体。其中 ,pB35 t SAG1以E35S驱动t SAG1,3′端携带NOS3′终止子序列 ,组成E35S/t SAG1/NOS3′操纵子结构单元。pB35E1 t SAG1含E35S和番茄果实特异性E8启动子核心序列E81.1双启动子驱动t SAG1,组成E35SE81.1/t SAG1/NOS3′操纵子结构单元。pBE2t SAG1以番茄果实特异性E8启动子全长E82 .2驱动t SAG1,组成E82 .2 /t SAG1/NOS3′操纵子结构单元。pB35 t SAG1、pB35E1 t SAG1、pBE2 t SAG1经酶切证实后 ,分别将其中E35S/t SAG1/NOS3′、E35S -E81.1/t SAG1/NOS3′、E82 .2 /t SAG1/NOS3′操纵子结构单元以HindⅢ单酶切连接方式亚克隆进pCAMBIA2 30 0质粒中 ,构建植物表达载体 pC35 t SAG1、pC35E1 t SAG1、pCE2 t SAG1,酶切鉴定。含三种启动子驱动t SAG1的植物表达载体转化根癌农杆菌LBA4 4 0 4感受态细胞后 ,
基金Project(200805032) supported by Grands from Scientific Research Program of Marine Public Welfare Industry of ChinaProject (DYXM-115-02-2-07) supported by China Ocean Mineral Resources R&D Association (COMRA)
文摘Culturable thermophilic microorganisms were enriched from samples collected from Lau Basin hydrothermal vents in artificial seawater medium at 45 ℃ and pH 7.0. Microbial diversities of the enriched communities were defined by performing a restriction fragment length polymorphism （RFLP） analysis of 16S rRNA gene sequences with enzymes MspI and Hin6 I. A total of 14 phylotypes have been detected by the RFLP patterns identified for 16S rRNA clone libraries of the enrichment. Analysis of sequences showed that at least four bacterial divisions presented in the clones libraries. The phyla Proteobacteria and Firmicutes were the most dominant groups. The majority of the sequences included in this analysis affiliated with Gamma Proteobacteria （71%） and Bacillus （23%）. Scanning electron micrographs revealed that there were abundant rod and coceoidal forms encased in sulphur and sodium chloride precipitate. These results revealed that there were a diversity of moderate thermophilic bacterial populations thrived in Lau Basin hydrothermal vents that were previously not detected by either molecular retrieval or strain purification techniques.
