根癌农杆菌(Agrobacterium tumefaciens)介导培育富含叶黄素的基因工程小球藻(Chlorella vulgaris)的研究

THE RESEARCH OF AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION OF GENE ENGINEERING CHLORELLA VULGARIS RICH IN LUTEIN

采用基因克隆技术构建双元载体pCAMBIA2301-idi,通过电转转入农杆菌LBA4404中。利用根癌农杆菌介导的转化方法,将pCAMBIA2301-idi质粒的T-DNA区转入小球藻,以G418抗性基因(NPTⅡ)作为筛选标记,筛选出阳性转化子。通过PCR扩增表明idi基因和NPTⅡ基因已经整合到小球藻基因组中。测定转化子的生物量,结果表明大部分转化子的生物量与野生型相似。测定转化子小球藻干粉的叶黄素含量,发现转化子叶黄素含量最高达到0.84mg/g,与野生型相比提高了30.95%。进一步分析藻液中叶黄素的产量,发现转化子的叶黄素产量最高达到1.98mg/L,比野生型提高了36.77%。

In this study, we used gene clone technology to construct binary vector pCAMBIA2301-idi, and transferred it into Agrobacterium tumefaciens LBA4404 by electroporation. The T-DNA region of pCAMBIA2301-idi was introduced into Chlorella vulgaris by A. tumefaciens-mediated transformation （ATMT）, using G418 resistance gene （NPT if） as selective marker which can be used to screen positive clones. The result of PCR showed that idi gene and NPTff gene were integrated into the genome of C. vulgaris. Further analysis found that the majority of the transformants displayed similar biomass compared with the wild type strain. While the highest lutein content of transformants reached 0.84mg/g, increasing by 30.95% compared with that of the wild type strain. And the result of analyzing lutein yield in the algal culture demonstrated that the highest lutein yield of transformants reached 1.98mg/L, increasing by 36.77% compared with that of the wild type strain.