食品科学

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生物合成γ-氨基丁酸酿酒酵母谷氨酸脱羧酶基因的克隆与表达

乌云达来1,2,张博润2,郭雪娜2,王肇悦2,*   

  1. 1.内蒙古农业大学食品科学与工程学院,内蒙古 呼和浩特 010018;2.中国科学院微生物研究所,北京 100101
  • 出版日期:2015-07-15 发布日期:2015-07-08

Molecular Cloning and Expression of Glutamic Acid Decarboxylase Gene from Saccharomyces cerevisiae for Biosynthesis of γ-Aminobutyric Acid

Wuyundalai1,2, ZHANG Borun2, GUO Xuena2, WANG Zhaoyue2,*   

  1. 1. College of Food Science and Engineering, Inner Mongolia Agricultural University, Hohhot 010018, China;
    2. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
  • Online:2015-07-15 Published:2015-07-08

摘要:

为了提高酵母菌的γ-氨基丁酸产量,本研究以十六烷基三甲基溴化铵法(hexadecyl trimethy ammoniumbromide,CTAB)提取的酿酒酵母28基因组DNA为模板,扩增得到序列长度为1 758 bp的GAD1基因,经比对与S. cerevisiae S288c的一致性达到98.58%,并设计引物扩增包括GAD1基因上游启动子及调控序列,下游终止子在内的全长基因序列,长度为3 490 bp。将全长基因序列克隆至高拷贝质粒pUG6,构建重组质粒p28。以菌株28作为出发菌株进行转化,经G418抗性筛选得到转化子,进一步经过聚合酶链式反应(polymerase chain reaction,PCR)实验验证,质粒提取及酶切验证,确证得到重组子DL28。经重组质粒p28的特性研究得知,重组质粒p28具有良好的遗传稳定性,无抗性传代培养10 次重组质粒不丢失。转化后进行酶活力测定,重组子DL28的谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)酶活力比菌株28提高73.8%。通过以上实验结果得出结论,GAD基因高表达菌株DL28具有更高的G418抗性和GAD酶活性。

关键词: 酿酒酵母菌, &gamma, -氨基丁酸, 谷氨酸脱羧酶, 克隆, 表达

Abstract:

Glutamic acid decarboxylase (GAD) is a rate-limiting enzyme for the biocatalysis of γ-aminobutyric acid
(GABA). Therefore, GAD gene is the key gene to regulate the production of GABA. In order to improve the productive
capacity of GABA in yeasts, in the present study, we used the CTAB method to purify S. cerevisiae 28 genomic DNA as the
template. A 1 758 bp gene fragment was amplified and sequenced as the GAD1 gene that had 98.58% homology with the
S. cerevisiae S288c gene. The primers were designed for amplifying the 3 490 bp sequence of GAD1 full-length gene,
including GAD1 gene upstream promoter, regulatory sequence and downstream terminator. The full-length gene sequence
was cloned into plasmid pUG6, to construct the recombinant plasmid p28. The strain 28 was used as the starting strain
for the conversion, and the transformant was gained by G418 resistance screening, and further verified by PCR, plasmid
extraction and double digest identification as the recombinant DL28. By exploring the characteristics of the recombinant
plasmid p28, we found that it had good genetic stability, and the recombinant plasmid was still stable after subculture for 10
generations without resistance training. The enzyme activity was determined after transformation, and the GAD activity of
recombinant DL28 was improved by 73.8% when compared with the strain 28. Therefore, GAD gene is highly expressed in
recombinant DL28, which also has higher G418 resistance.

Key words: S. cerevisiae, γ-aminobutyric acid, glutamic acid decarboxylase, cloning, expression

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