同源重组敲除三角褐指藻基因的研究

为探索应用基因敲除技术研究三角褐指藻基因功能的研究体系,本文以三角褐指藻甘油激酶基因作为靶基因,构建了同源重组基因敲除载体,利用微弹轰击法将该载体成功转化至三角褐指藻中,经100μg/mL Zeocin筛选和PCR验证获得了34个阳性转基因藻株;并进一步对三角褐指藻甘油激酶基因敲除转基因藻株的甘油激酶表达量和生长两方面进行分析,结果显示胞外甘油兼养不影响转基因藻细胞生长,甘油激酶不表达或表达量降低.本文通过构建敲除载体,完成了遗传转化,筛选获得阳性转基因藻,并进一步研究了转基因藻的性状,最终建立了应用同源重组基因敲除技术靶向研究三角褐指藻基因功能的研究体系.

The research of gene knockout via homologous recombination in Phaeodactylum tricornutum

Phaeodactylum tricornutum is model organism for marine diatom. It is also one of the most popular energy microalgae. The genome sequence of P. tricornutum had been completed. But, the gene function analysis and gene regulation research of P. tricornutum need further exploration. In this paper, we constructed the glycerol kinase gene knockout vector for P. tricornutum, and successfully converted the vector to wild type P. tricornutum by particle bombardment. After screened, 34 transformations were obtained. The analysis of cell growth showed that extracellular glycerin had no effect on the cell growth of the transformats. Detected by western blot, the results presented that the expression of glycerol kinase were repressed or reduced in transformants. In short, we explored a knockout system for further gene function research in P. tricornutum.