过表达purA基因对腺苷积累的影响

Effect of purA Gene Overexpression on Adenosine Accumulation

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.