利用PTVA法提高超氧化物歧化酶基因在毕赤酵母中的表达  

Improvement of SOD gene expression level in Pichia pastoris by PTVA

作　　者：李潇楠[1,2] 王爽[1] 王琦[1] 

机构地区：[1]中国农业大学农学与生物技术学院/农业部植物病理学重点开放实验室,北京100193 [2]全国农业技术推广服务中心,北京100125

出　　处：《中国农业大学学报》2012年第3期88-93,共6页Journal of China Agricultural University

基　　金：国家科技支撑计划资助项目(2007BAD57B02)

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150U/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。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.

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 

分 类 号：Q344.13[生物学—遗传学]