all 1691基因表达及铁元素对鱼腥藻生长的影响

Expression of the gene all 1691 and effect of Fe^(3+) on the growth of Anabaenasp.strain PCC7120

为实现FurA的表达,探究铁元素对藻生长的影响,首先运用Primer5.0设计引物,克隆出鱼腥藻PCC7120染色体上all1691(furA)基因片段;构建含组氨酸融合表达标签和T7启动子标签的表达载体.IPTG诱导FurA蛋白表达.然后设计不同Fe3+浓度梯度培养基,利用SDS-PAGE检测高、中、低Fe3+浓度培养液的藻细胞总蛋白,考马斯亮蓝g-250法测定蛋白含量,并用Trizol法提取不同Fe3+浓度培养的藻细胞总RNA.结果表明,获得纯化的all 1691克隆片段,大小为456bp,pET-1691重组蛋白在1mmol/L的IPTG诱导下,于37℃下摇床培养15h得到成功表达;低浓度Fe3+促进藻生长,高浓度Fe3+抑制藻生长;Fe3+浓度为2.0mg/L时rRNA位置与亮度清晰可见,而缺铁培养的藻生长几乎停滞,转录和翻译水平都很低.

To realize the expression of FurA and explore the effect of Iron on the growth of Anabaena sp. strain PCC7120. First the primers were designed by Primer 5.0. The chromosomal gene all 1691 （furA） of Anabaena sp. strain PCC7120 was cloned ; Expression vector was constructed containing L-Histidine fusion expression tag and T7 promoter tag. The expression of FurA protein was induced by IPTG. Then culture mediums with different concentration gradient （high.medium and low）of Fe^3＋ were devised to detect the total protein of Anabaena cells by SDS-PAGE. Protein content was determined by Coomassie brillant blue g-250method. And total RNA of algae cultured with different concentration gradient of Fe3＋ were extracted via the Trizol method. The results showed that the purified all 1691 fragment was obtained which size of 456bp. The pET-1691 of recombinant protein was expressed successfully in vitro under the condition that induction of 1 mmol/L IPTG and 37℃ shaking culture of 15 h. Low concentration of Fe^3＋ promoted the growth algae, while high concentration of Fe^3＋ inhibited the growth of algae. The position and brightness of rRNA were clearly visible when the concentration of Fe^3＋ was 2.0 mg/L. However the Anabaena growth was almost stalled and the level of transcription and translation were both very low with when cultured iron deficiency.