【作者】 张小波；

【导师】 夏焕章；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2002， 硕士

【摘要】 为了研究黑暗链霉菌安普霉素生物合成基因，首先通过鸟枪克隆的方法克隆其安普霉素抗性基因。提取黑暗链霉菌H6总DNA，经Sau3AI不完全酶切、凝胶电泳，收集6～15kb片段连接到载体pIJ702。连接液转化标准宿主菌S.lividans TK24原生质体，最终得到6个可以在安普霉素平板上生长的阳性克隆。阳性克隆中重组质粒pSYX1含有12kb黑暗链霉菌的DNA，对12kb片段进一步分析将安普霉素抗性基因定位在1.5kb SphI-KpnI片段上。 抗性基因上游2.0kb BamHI-SphI片段测序表明在该片段中有一个完整的ORF，位于877～1995bp之间，编码373个氨基酸。在这个ORF中，碱基组成为109A+388C+489G+136T，G+C含量为78.2％，密码子第三位碱基是G+C的占96.3％，符合链霉菌基因的特点。同源比较分析没有发现同源序列，推测这一序列可能是一未知的新基因，命名为aprA。 将克隆到的链霉菌DNA2.0kb片段以及报告基因ermE、xylE插入到具有oriT的大肠杆菌—链霉菌穿梭质粒pHZ132中构建接合转移质粒pZXB014，并将其转入大肠杆菌ET12567(pUZ8002)中，获得供体菌ET12567(pUZ8002，pZXB014)。通过接合转移的方法将质粒pZXB014导入黑暗链霉菌H6中，筛选基因发生重组的菌株。PCR实验证明基因重组菌株中接合转移质粒同源整合到黑暗链霉菌H6的染色体上。基因重组菌株不能合成安普霉素，证明克隆到的aprA基因参与黑暗链霉菌安普霉素的合成，同时也证明在黑暗链霉菌中安普霉沈阳药科人学颀上学位论文 摘要素生物合成基因与安普霉素抗性基因是连锁存在的。更多还原

【Abstract】 To study the biosynthetics genes of apramycin in Streptomyces tenebrarius, the apramycin resistant gene was isolated by gunshot cloning firstly. S.tenebrarius chromosomal DNA , partially digested with Sau3Al to yield fragments of 6 ~ 15kb, was ligated with vector pIJ702. The ligation mixture was used to transform S.lividans TK24 protoplasts. Six colonies capable of growing in the presence of apramycin were isolated. Plasmid pSYXl, containing a 12kb insert of S.tenebrarius DNA, was isolated from one of six colonies. Apramycin resistant gene was located in 1.5-kb Sphl-Kpnl fragment of plasmid pSYXl though further studies.The 2.0-kb BamHl-Sphl fragment, upstream fragment of the apramycin resistant gene, was sequenced. Analysis of the nt sequence revealed that it contained an ORF from nt positions 877 to 1995. Composed of 109A, 388C, 489G and 136T, the ORF encoded a protein of 373 amino acids. The G+C percentage of this ORF was 78.2% and that of the third base of the codons was 96.3%. No homologous sequence was found by comparing it with the sequences obtained from the net, so that the cloned gene was supposed to be an unknown new gene, designated as oprA.A fragment, containing 2.0kb cloned 5. tenebrarius DNA and reported genes of ermE and xylE , was inserted in plasmid pHZ132 ( anE.coli-Streptomyces shuttle plasmid incorporating oriT from RK2) to construct disruption plasmid pZXB0l4. The plasmid was transformed into E.coli ET12567(pUZ8002) to construct recombinant E.coli ET12867(pUZ8002, pZXB014 ). Recombinant S.tenebrarius was obtained by conjugation E.coli ET12867(pUZ8002, pZXB014) with S.tenebrarius H6 to scan strains of which plasmids integrated into S.tenebrarius H6 chromosomal DNA. The gene recombinant strain NO.42 can’t generate ampramycin, which indicated that the cloned gene is involved in apramycin biosynthesis in S.tenebrarius. It was also proved that the biosynthestic genes of apramycin was linked to the apramycin resistant gene in S.tenebrarius.更多还原