摘要
采用PCR的方法从产甘油假丝酵母(Candida glycerinogenes)中扩增出3-磷酸甘油脱氢酶的编码基因及侧翼序列CgGPD1,分别构建了含不同CgGPD1拷贝数的根癌农杆菌双元载体pCAM3300?zeocin-CgGPD1(Ⅰ)、pCAM3300-zeocin-CgGPD1-CgGPD1(Ⅱ)和pCAM3300-zeocin-Cg GPD1-CgGPD1-CgGPD1(Ⅲ)。在大肠杆菌JM109中,研究了其在不同质量浓度NaCl、葡萄糖胁迫下表达情况。结果表明,在大肠杆菌中GPDH的活性随着NaCl、葡萄糖质量浓度的升高而增加。当NaCl质量浓度达到2.5 g/dL时,GPDH的酶活比在0.5 g/dL NaCl下平均提高31.2%;当葡萄糖质量浓度提高至10 g/dL时,GPDH的酶活比2 g/dL葡萄糖下平均提高31.8%;在相同的NaCl、葡萄糖质量浓度下,GPDH的活性随着CgGPD1拷贝数的增加而升高,JM109(Ⅱ)比JM109(Ⅰ)的GPDH酶活平均提高8.2%,JM109(Ⅲ)比JM109(Ⅱ)的GPDH酶活平均提高9.9%。以上结果表明,在大肠杆菌中CgGPD1基因的表达同样受渗透压胁迫调节。
Candida glycerinogenes, a novel osmotolerant yeast, used for industrial-scale fermentation and glycerol-3 phosphate dehydrogenase, the key enzyme of glycerol synthesis, was induced by osmotic stress. In this manuscrigt, we the gene of glycerol-3-phosphate dehydrogenase GPD1 from C. glycerinogenes was cloned and a binary vectors with different copies of CgGPDI： pCAM3330-zeocireCgGPD1 （Ⅰ）, pCAM3330-zeocircCgGPD1-CgGPD1 （Ⅱ） and pCAM3330-zeocirt-CgGPD1-CgGPD1-CgGPD1 （Ⅲ） were constructed. The vectors were transformed into Escherichia coli JM109. The expression of GPDH from the transformants harboring different copies of CgGPD1 under different concentration of NaCI and glucose were determined. The results showed that the activity of glycerol-3-phosphate dehydrogenase was increased about 31. 5% when the concentration of NaCl or glucose was 2.5% or 10%, respectively. The activities of glycerol-3-phosphate dehydrogenase of JM109（Ⅱ） and JM109（Ⅲ） were 8.2% and 9.9% higher than that of JM109（Ⅰ） and JM109（Ⅱ）, respectively. The results indicated that the expression of CgGPD1 was also induced by osmotic stress in E. coli JM109.
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2008年第5期50-56,共7页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(30570142
20676053)
国家863计划项目(2006AA020103)
江苏省青年科技创新人才基金项目(BK2006504)
