发菜(Nostoc flagelliforme)是一种陆生蓝藻,前期转录组测序发现糖基转移酶基因转录水平较正常上调2.29倍.以发菜基因组为模板克隆糖基转移酶基因,获得长度为1290 bp的核酸序列.通过生物信息学分析表明,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2、9、18,蛋白质分子量为47.54 kDa,等电点为9.33.正负电荷氨基酸残基数分别为61和51.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 mM IPTG在16℃下诱导表达20 h后,获得预期大小重组蛋白(47.54 kDa).研究首次成功克隆得到发菜糖基转移酶基因,并构建重组质粒于大肠杆菌中高效表达,实验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,也为今后糖基转移酶基因工程菌株的构建提供理论依据.
Gene cloning and prokaryotic expression of glycosyl transferase from Nostoc f lagelli f orme
Nostoc flagelliforme is a terrestrial cyanobacteria ,It was found in the previous work that the transcription level of gene encoded for glycosyl transferase increased 2 .29 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 tem-plate to clone the gene encoded for glycosyl transferase .The sequence with the size of 1290 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 2 ,9 and 18 ,respectively .The molecular weight of the protein is 47 .54 kDa and the isoelectric point is 9 .33 .The amino acid residues with positive and negative charges are 61 and 51 re-spectively .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 (47 .54 kDa) was ob-tained after 20 hours of induction with 1 mM IPTG in 0 .8 OD value at 16 degree .In this work ,the gene for glycosyl transferase from Nostoc flagelliforme was successfully cloned and expressed in escherichia coli for the first time .The result may lay a foundation for fur-ther study on molecular mechanism of polysaccharide metabolism from N .flagelliforme and provide theoretical basis for the construction of engineered strain for glycosyl transferase in the future .
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