【摘要】 以“连续延伸 PCR基因合成法”(姚泉洪 1999) ,按植物偏爱密码子 ,合成了全长4 4 1bp的透明颤菌血红蛋白 (Vitreoscilla hemoglobin VHb)基因。此方法不需用连接酶 ,而用高保真 DNA聚合酶 pwo及 7个长度为 90 bp左右的长寡核苷酸引物 ,经一次 PCR反应即完成了全长为 4 4 1bp基因合成。 PCR产物经 Bam HI和 Sac I酶切 ,克隆入 p Bluescript载体 ,合成的 VHb基因经克隆和测序证实与设计一致。与报道的 VHb基因相比 ,核苷酸改动了 98个。G+C含量由原来的 4 5.3%提高到 4 7.8%。将该基因克隆入庆大霉素抗性基因启动子后 ,构建表达载体 p YP2 0 0 1h(VHbp)。将其转入大肠杆菌菌株 DH5α,在氧胁迫、缺乏、充足等三种情况下 ,绘制其生长曲线 ,探讨了合成 VHb基因在原核生物中的效应。结果显示 ,合成的 VHb基因在氧胁迫条件下能加快细菌的生长 ,增加产量更多还原

【Abstract】 Vitreoscilla hemoglobin (VHb) gene was designed and synthesized using the successive PCR technique (Yao QH 1999) for the optimal expression in plant. The 441 bp VHb gene synthesis does not rely on the DNA ligase but relies on high fidelity DNA polymerase pwo. The VHb gene was assembled through one round of PCR from a total of only seven 90bp oligos. The synthesized gene was digested with BamHI and SacI , and inserted into the pBluescript vector. Finally, the nucleotide sequence of synthesized VHb gene was determined. The result indicates that the nucleotide sequence of the VHb gene is the same as the sequence designed. 98 nucleotides are changed in the synthesized VHb gene and the G+C content was increased from 45.3% to 48.7%.The gene was inserted into a prokaryotic expression vector controlled by Gm r promotor and then transferred into E.coli strain DH5α. The growth curves of VHbp and CK strain were drawn in oxygen stress, poor oxygen and full oxygen conditions respectively,then the effect of VHbp in prokaryotic organism was discussed. The result demonstrated that the synthesized VHbp gene can enhance the growth speed of bacteria and increase the yield of bacteria.更多还原