过表达<em>purA</em>基因对腺苷积累的影响

中国生物工程杂志

2011, Vol. 31

Issue (12): 22-26

研究报告

过表达purA基因对腺苷积累的影响

张洋, 杜姗姗, 谢希贤, 徐庆阳, 陈宁

天津科技大学生物工程学院天津 300457

Effect of purA Gene Overexpression on Adenosine Accumulation

ZHANG Yang, DU Shan-shan, XIE Xi-xan, XU Qing-yang, CHEN Ning

School of Biological Engineering, Tianjin University of Science & Technology, Tianjin 300457, China

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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-AG^r+SG^r)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5 L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。

关键词： 腺苷; 腺苷酸琥珀酸合成酶; 枯草芽孢杆菌

Abstract:

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 pBE43∷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.

Key words: Adenosine Adenylosuccinate synthetase Bacillus subtilis

收稿日期: 2011-09-20 出版日期: 2011-12-25

ZTFLH:

Q789

通讯作者: 陈宁,电子信箱:ningch@tust.edu.cn E-mail: ningch@tust.edu.cn

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引用本文:

张洋, 杜姗姗, 谢希贤, 徐庆阳, 陈宁. 过表达purA基因对腺苷积累的影响[J]. 中国生物工程杂志, 2011, 31(12): 22-26.

ZHANG Yang, DU Shan-shan, XIE Xi-xan, XU Qing-yang, CHEN Ning. Effect of purA Gene Overexpression on Adenosine Accumulation. China Biotechnology, 2011, 31(12): 22-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I12/22

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