鱼腥藻铁调蛋白基因(alr0957)表达和Fe^(3+)对藻生长的影响

Expressing of the Gene fur( alr0957) and Effect of Fe^(3 +) on the growth of Anabaena sp. strain PCC7120

根据CyanoBase提供的鱼腥藻PCC7120 FurC基因(alr0957)序列信息设计特异性引物,用降落PCR法从染色体DNA中扩增得约450 bp目的片段.运用TA克隆将其连接到pMD18-T载体上,经筛选测序获得阳性重组质粒.再经过双酶切、纯化FurC基因,连接原核表达载体pET-28a(+),并转化表达菌株BL21和IPTG诱导表达.经测序鉴定的阳性菌株中的FurC,运用SDS-PAGE检测重组蛋白,并利用镍柱层析法纯化目的蛋白.由藻细胞密度、叶绿素a含量、可溶性糖含量等改变探讨不同Fe3+浓度对藻类生长的影响.结果表明:在37℃经1 mmol/L IPTG诱导18 h,成功表达了分子量约为19000的融合蛋白.小于0.5mg/L Fe3+促进藻生长,大于0.9 mg/L Fe3+抑制藻生长,最适藻生长Fe3+浓度为0.5~0.9 mg/L.

The specific primers were designed according to Anabaena PCC 7120 FurC gene （alr0957） sequence provided by CyanoBase .A 450 bp DNA target fragment was amplified from chromosome by touchdown PCR and was connected to pMD 18-T vector by TA cloning .The recombinant plasmid was obtained by screening and sequencing .Then it was connected to the prokaryotic expression vector pET-28a （＋） after double digestion and purification and transformed into the expression strain BL21.IPTG induced FurC from positive strains was identified by sequencing .The recombinant proteins were then detected through SDS-PAGE and purified by nickel column chromatography .Finally the effect of Fe3＋concentration on algae growth was explored with regard to algal cell density , chlorophyll content , soluble sugar content , etc.The results showed that fusion protein with molecular weight of 19000 was successfully expressed under 37℃, 1 mmol/L IPTG induction for 18 h.In addition, an Fe3＋concentration less than 0.5mg/L promoted the algal growth while a concentration more than 0.9 mg/L inhibited the growth of algae .So the optimum concentration of Fe 3＋for the algal growth is in the range of 0.5 -0.9 mg /L.