食品与发酵工业2018，Vol.44Issue(1)：31-36,6.DOI:10.13995/j.cnki.11-1802/ts.015667
发菜中GDP-甘露糖4,6-脱水酶基因的克隆及原核表达
Gene cloning and prokaryotic expression of GDP-mannose 4,6-dehydratase from Nostoc flagelliforme
摘要
Abstract
Nostocflagelliforme is a terrestrial cyanobacteria with high stress-tolerance.It is able to lives in harsh environment.The extracellular polysaccharide produced by Nostocflagelliforme is edible and and proved to have high medicinal value.Previous work had found that using RNA-Seq method,the transcription level of gene encoded for the GDP-mannose 4,6-dehydratase was by 2.18 times higher in Nostoc flagelliforme cultured under high salt concentration than the control.The primer was designed based on the transcriptome sequencing result and the homologous blast.Then,Nostoc flagelliforme genome was utilized as template to clone the GDP-4,6-mannose dehydratase encoding gene.The sequence with the size of 1080 bp was successfully obtained.According to the bioinformatical analysis,this gene was highly conserved;the translated protein was hydrophilic,and its structure was mainly consisted of random coiling and alpha helix.There were 13,15 and 17 phosphorylation sites of tyrosine,threonine,and serine respectively.The molecular weight of the protein was 41.08 kDa and the isoetectric point was at 5.73.The amino acid residues with positive and negative charges were 38 and 46,respectively.The recombinant plasmid was constructed by inserting the gene into the plasmid Pet28a,which transformed into Escherichia coli BL21 for prokaryotic expression.The expected size of recombinant protein (41.08 kDa) was obtained after 20 hours of induction with 1 mmol/L IPTG at 0.8OD under 16 ℃.In this work,the gene for GDP-4,6-mannose dehydratase from Nostocflagelliforme was successfully cloned and expressed in Escherichia coli for the first time.The result laid a foundation for further study to understand polysaccharide metabolism of N.flagelliforme,and provided theoretical basis for the construction of engineered strain for GDP-4,6-mannose dehydratase in the future.关键词
发菜/盐胁迫/GDP-甘露糖/4,6-脱水酶基因/克隆/原核表达Key words
Nostoc flagelliforme/salt stress/GDP-4,6-mannose dehydratase gene/clone/prokaryotic expression引用本文复制引用
蔡国强,陈雪峰,范华,刘欢,赵圆圆..发菜中GDP-甘露糖4,6-脱水酶基因的克隆及原核表达[J].食品与发酵工业,2018,44(1):31-36,6.基金项目
国家自然科学基金委员会生命科学部2014年度青年基金项目(31401633) （31401633）
西安市发展和改革委员会西安市微生物药物工程实验室项目 （）
(2014)陕西省重点研发计划项目(2017NY-146) （2014）
