基因枪法建立紫菜丝状体遗传转化模式

Establishment of a concocelis-mediated transformation system for Porphyra by biolistic PDS-1000/He

以坛紫菜和条斑紫菜的自由丝状体为材料,利用基因枪法分别转化CaMV35S、SV40、FCP、Amt、Ubi 5种启动子与报告基因组合,转化后48h进行原位组织化学染色检测,首次针对重要经济红藻紫菜的自由丝状体建立并优化遗传转化技术,为紫菜研究提供了新工具。结果显示,SV40启动子可以驱动laeZ报告基因(编码β-半乳糖苷酶)在紫菜丝状体中的瞬间表达,空白与阴性对照未检测到本底;其它4种启动子未检测到基因表达。进一步优化实验发现,在可裂膜650psi、轰击距离6cm下获得最高转化效率为8.0×10^(-5),经定量检测与双因子方差分析,是最佳转化参数,提示基因枪参数对外源基因转化效率具显著影响。

Using a biolistic PDS 1000/He system, free-living conchocelis of Porphyra haitanensis （Rhodophyta） and P. yezoensis are bombarded respectively to test five heterologous promoter-reporter gene cassettes under different biolistic parameters. According to the results of in situ histochemical staining 48 hours after transformation, this is the first report on optimization of Porphyra concocelis-mediated transformation. The established transient expression technique could serve as a new tool for Porphyra research in the concocelis stage, and be ready to set up a stable expression system. It is showed that only the SV40 promoter could drive the transient expression of lacZ reporter gene without background in either blank control or negative control, whereas CaMV35S, FCP, Amt and Ubi promoters all failed to be recognized effectively by Porphyra concocelis. The highest transformation efficiency of SV40-lacZ is obtained as 8.0×10^-5, suggesting that 650 psi rupture-disc pressure and 6cm of bombardment distance are optimal biolistic parameters for Porphyra concocelis, and it is also proved by quantitative determination to lacZ expression.