发菜(Nostoc flagelliforme)，是一种陆生蓝藻，生长环境恶劣，具很强的抗逆性，其分泌的胞外多糖具有极高的食用和药用价值,但其合成机制尚不清楚。前期工作采用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1080bp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，蛋白质分子量为41.08 kDa，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℃下诱导表达20小时后，获得预期大小重组蛋白(41.08 kDa)。本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，研究结果为进一步研究发菜多糖代谢分子调控机制奠定了基础，也为今后GDP-甘露糖4,6-脱水酶基因工程菌株的构建提供理论依据。

Nostoc flagelliforme is a terrestrial cyanobacteria with stress-tolerance, which lives in poor condition. And the extracellular polysaccharide produced by Nostoc flagelliforme is proved to be with high edible and medicinal value. It was found in the previous work that the transcription level of gene encoded for GDP-mannose 4,6-dehydratase increased 2.18 times in Nostoc flagelliforme cultured under high salt concentration contrast to the control by RNA-Seq technology. Based on the transcriptome sequencing result and the homologous blast, the primer was designed  and the genome of Nostoc flagelliforme was utilized as template to clone the gene encoded for GDP-4,6-mannose dehydratase. The sequence with the size of 1080 bp was successfully obtained. According to the bioinformatical analysis, this gene was highly conserved, and the translated protein is hydrophilic and mainly consists of random coiling and alpha helix. The number of phosphorylation sites of tyrosine, threonine, and serine was 13, 15, and 17, respectively. The molecular weight of the protein is 41.08 kDa and the isoelectric point is 5.73.The amino acid residues with positive and negative charges are 38 and 46 respectively. Then, the recombinant expression plasmid was constructed by inserting the gene into the plasmid, Pet28a and successfully introduced into the competent 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 in 0.8 OD value at 16 degree. In this work, the gene for GDP-4,6-mannose dehydratase from Nostoc flagelliforme was successfully cloned and expressed in Escherichia coli for the first time. The result may lay a foundation for further study on molecular mechanism of polysaccharide metabolism from N. flagelliforme and provide theoretical basis for the construction of engineered strain for GDP-4,6-mannose dehydratase in the future.