摘要：
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 °C.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.%前期采用RNA-Seq技术对高盐胁迫下的发菜样品进行转录组测序,发现GDP-甘露糖4,6-脱水酶基因转录水平较正常培养条件上调2.18倍.基于转录组测序结果,通过同源相比对分析设计引物,以发菜基因组为模板克隆GDP-甘露糖4,6-脱水酶基因,获得长度为1 080 bp的核酸序列.通过生物信息学分析表明,该基因具有较高保守性,蛋白质二级结构主要构成方式为随机卷曲和α螺旋,是亲水性蛋白,酪氨酸、苏氨酸、丝氨酸磷酸化位点个数分别为13、15、17,蛋白质分子质量为41.08 ku,等电点为5.73.正负电荷氨基酸残基数分别为38和46.随后,将该基因插入质粒pET-28a中成功构建重组表达质粒,然后转化至BL21大肠杆菌感受态中进行原核表达.在OD值为0.8时,用1 mmol/L IPTG在16°C下诱导表达20 h后,获得预期大小重组蛋白(41.08 ku).该研究首次成功克隆得到发菜GDP-甘露糖4,6-脱水酶基因,并成功构建重组表达质粒于大肠杆菌高效表达.
