含铁蛋白FAO1融合基因的衣藻核转化表达和分析.pdfVIP

中国农业科学 2013,46(21):4515-4522 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2013.21.014 含铁蛋白FAO1 融合基因的衣藻核转化表达和分析 李明泽，程 奇 （中国农业科学院生物技术研究所，北京100081） 摘要：【目的】fao1 编码Candida cloacae 中一种含铁辅基的长链脂肪醇氧化酶。通过构建该基因的衣藻核 转化表达载体进行转化，根据表达蛋白的生物活性判断fao1 能否充分利用衣藻叶绿体中的铁元素行使功能；同时 也为需铁固氮酶基因的研究提供方向。【方法】通过PCR 把PsaD 信号肽、fao1、His-tag 标签融合，连接到pDBle 载体上；采用玻璃珠转化法，将重组子导入莱茵衣藻（CW15）中；经博来霉素筛选后，获得转基因植株。运用PCR 和RT-PCR 法检测了转化衣藻，并且用亲和层析柱纯化蛋白，同时进行FAO1 活性检测。【结果】重组质粒测序完全 正确；PCR 和RT-PCR 检测转化衣藻基因组DNA 和RNA，扩增片段与预期相符；转化衣藻FAO1 蛋白纯化洗脱液使 ABTS （2,2′-联氨-双（3-乙基苯并噻唑啉-6-磺酸）二胺盐）底物反应变色。【结论】成功构建了信号肽、FAO1 和His-tag 融合基因的衣藻核转化表达载体，重组质粒已整合到莱茵衣藻基因组中，并成功转录，纯化的蛋白检 测有活性。 关键词：长链脂肪醇氧化酶；信号肽；His-tag 标签；衣藻核转化；RT-PCR Expression and Analysis of Candida cloacae Long-Chain Fatty Alcohol Oxidase FAO1 by Nuclear Expression Vector in Chlamydomonas reinhardtii LI Ming-ze , CHENG Qi (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081) Abstract: 【Objective 】fao 1 encods a iron-containing long-chain fatty alcohol oxidase in C.cloacae. By constructing the nuclear expression vector, according to the biological activity of protein we can judg e whether fao1 can take full advantage of iron in Chlamydomonas reinhardtii chloroplast. Meanwhile it will provide a new direction for researches on the gene of iron-containing proteins. 【Method 】The PsaD transit peptide gene(TP), C. cloacae fatty-alcohol oxidase (fao1) gene and His-tag were cloned separately by polymerase chain reaction (PCR). The 3-piece fusion gene TFHis was inserted into pDBle. This construct was transforme