| 摘要: |
| 为得到高表达超氧化物歧化酶毕赤酵母工程菌株,促进SOD的产业化生产,将来源于蜡样芽孢杆菌M22菌株的Mn-SOD-2基因转入毕赤酵母GS115中,构建了表达工程菌株YS 1~100。采用PTVA法(Posttransformational vector amplification)进行拷贝数扩增处理。建立了SOD重组菌株MMH(Minimal Methanol+Histidine)平板简易检测法,使用该法对转化子进行初筛,并用荧光定量PCR(Fluorescence quantitative PCR)法检测部分重组酵母工程菌中外源SOD基因的拷贝数。结果表明:试验中得到80株高抗Zeocin的菌株YSP 1~80,经MMH平板法筛选得到菌株YSP14、YSP30、YSP48和YSP54的SOD酶活性明显高于处理前;菌株YSP54的SOD基因的拷贝数达到8个拷贝,而处理前仅为1个拷贝;Native-Page分析与NBT酶活性测定显示菌株YSP54发酵上清液中的SOD酶活性达到150 U/mL,为处理前的2倍。提高毕赤酵母中外源SOD基因的拷贝数可以相应提高SOD的酶活。 |
| 关键词: 毕赤酵母 超氧化物歧化酶 质粒转化后扩增法 基因拷贝数 MMH平板检测法 |
| DOI：10.11841/j.issn.1007-4333.2012.03.014 |
| 投稿时间：2011-12-14 |
| 基金项目:国家科技支撑计划资助项目(2007BAD57B02) |
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| Improvement of SOD gene expression level in Pichia pastoris by PTVA |
|
LI Xiao-nan1,2, WANG Shuang1, WANG Qi1
|
| (1.College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China;2.National Agro-technology Extensive and Service Center,Beijing 100125,China) |
| Abstract: |
| Mn-SOD-2 gene of Bacillus cereus M22 strain was integrated into the genome of Pichia pastoris GS115 in order to obtain the high-level expression transformants (YS1-YS100) of Mn-SOD-2 gene.The PTVA (Posttransformational vector amplification) method was used to increase the copy number of SOD gene.The transformants with high activity of SOD were preliminarily filtrated by MMH (Minimal Methanol+Histidine) plate test method.The SOD gene copy number of some positive transformants was then detected by FQ-PCR.The obtained transformants of YSP1-YSP80 all had high resistance to zeocin.The SOD activity of YSP14,YSP30,YSP48,YSP54 were significantly higher than that of YS.The SOD gene copy number of YSP54 reached 8 copies,but one copy in YS54.The analysis of Native-PAGE and NBT test showed that the maximum activity of the YSP54 was 150 U/mL,nearly twice as high as that of the original strain.It could be concluded that the increase of Mn-SOD-2 gene copy number in GS115 lead to higher activity of SOD. |
| Key words: superoxide dismutase Pichia pastoris PTVA gene copy number MMH plate test method |
