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。
[Objective] faol 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 judge whetherfaol 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 （faol） 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 transformed into C. reinhardtii CW15 by glass beads method. The transgenic Chlamydomonas was obtained under the selection of zeocin and they were confirmed positive by PCR amplification and RT-PCR. The activity of the purified recombinant protein was tested by a color-linked assay. [Result] The correctness of the constructed pDBIe-TFHis was confirmed by DNA sequencing. PCR and RT-PCR analysis of the genomic DNA and RNA from transgenic Chlamydomonas showed that the recombinant plasmid has been integrated into C. reinhardtii CW15 genome and transcribed successfully. Protein purification elution made substrate reaction turn blue. [Conclusion] In this study, faol-bearing nuclear expression vector pDBle-TFHis was correctly constructed and integrated into C. reinhardtii CW15 genome andfaol transcript was detected successfully. The recombinant protein was purified by an affinity column and the activity was tested.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第21期4515-4522,共8页
Scientia Agricultura Sinica
基金
国家科技部"973"项目(2010CB126500)
国家自然科学基金(NSFC31070720)
