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过表达purA基因对腺苷积累的影响 被引量:3

Effect of purA Gene Overexpression on Adenosine Accumulation
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摘要 利用穿梭质粒pBE43,在腺苷产生菌枯草芽孢杆菌Bacillus subtilis XGL(Xan-+Deam-+8-AGr+SGr)中过表达腺苷生物合成途径中关键酶腺苷酸琥珀酸合成酶(ADSS)的基因purA,研究其对腺苷积累的影响。以腺苷产生菌XGL基因组为模板,扩增出purA基因,将其连接到穿梭载体pBE43上,获得重组质粒pBE43∶∶purA。将重组质粒转化入XGL中,构建新的产生菌XGL-SY。通过逆转录PCR和定量PCR测定purA基因在腺苷产生菌XGL和XGL-SY中的转录量,并利用5L罐发酵实验测定发酵参数的变化。分析发酵对数期的基因转录发现,在添加质粒后,purA基因的转录量提高了约9倍。上罐实验表明改造后的产生菌XGL-SY,菌体生长虽然受到一定的影响,但在相同发酵周期内腺苷产量提高了10.8%。研究结果表明在腺苷产生菌中过表达purA基因可以促进腺苷的积累,这为腺苷工程菌的进一步改造奠定了基础。 Effect of purA gene overexpression on adenosine accumulation was studied in industrial strain Bacillus subtilis XGL( Xan- + Deam- + 8-AG^r + SG^r). PurA gene was cloned from XGL's chromosome by PCR and ligated into the shuttle vector pBE43 which contained P43 promoter. Recombinant plasmid pBFA3: :purA was transformed into XGL by electroporation. Reverse transcription PCR and quantitative PCR were used for measuring the transcription of purA. Fermentation parameters were determined in 5 L fermenter. Expression analysis showed that the purA in XGL-SY expressed as 9 times many as that in XGL. Cell growth was dragged a little. However, the production of adenosine was 10. 8% higher than before. In conclusion, the overexpression of purA can promote further accumulation of adenosine, which made a foundation to further genetic engineering strain construction.
出处 《中国生物工程杂志》 CAS CSCD 北大核心 2011年第12期22-26,共5页 China Biotechnology
关键词 腺苷 腺苷酸琥珀酸合成酶 枯草芽孢杆菌 Adenosine Adenylosuccinate synthetase Bacillus subtilis
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