等鞭金藻的电转化体系

以等鞭金藻(Isochrysis sp.CCMM5001)为材料,探索了影响其电转化的主要因素,建立了等鞭金藻电转化体系。结果表明,真空抽滤法收集藻体获得的细胞存活率明显高于离心收集法;藻体收集的最适缓冲液为1.0 mmol/L HEPES(pH7.5)(含0.5 mol/L蔗糖);相同电场强度下,对数早期的藻细胞存活率最高,早、中、晚期的半致死电场强度分别为0.82～1.63 kV/cm、0.79～1.34 kV/cm、0.54～1.18 kV/cm;最佳脉冲时间为5 ms;冰浴时间为10 min。通过电击转化法向球等鞭金藻中导入质粒pCAMBIA2301,并对转基因球等鞭金藻进行GUS组织化学染色和GUS荧光定量分析,检测pCaMV 35S启动下的GUS报告基因的表达。得到转基因球等鞭金藻的GUS活性为0.52 nmolMU/(min.mg)。通过PCR方法检测固体平板上的单藻落,结果显示转化藻株能够检测到nptII基因。本研究建立了等鞭金藻的电转化体系,为外源基因的导入及其功能的研究奠定了基础。

Electric shock transformation method of Isochrysis sp.

The research is to explore the main factors about the transforming the exogenous DNA into Isochrysis sp.with electric shock transformation method,such as the growth phase of Isochrysis sp.,electric field strength,pulse time and ice bath time.The power conversion system was established.The results showed that the survival rate of cell obtained by the vacuum leaching method was significantly higher than by the centrifugation method.And the highest survival rate of Isochrsis sp.is with the buffer at the concentration of 1.0 mmoL/L HEPES(pH 7.5,containing 0.5 mol/L sucrose).Under the same electric field intensity,the survival rate of cell is the highest in the early logarithmic.According to the survival rate must be between 40% and 60%,the half lethal field strengths of Isochrysis sp.in early,middle and late growth phase were 0.82~1.63 kV/cm,0.79~1.34 kV/cm and 0.54~1.18 kV/cm,respectively.The optimum pulse time and ice bath time were 5 ms and 10 min,respectively.Transforming the exogenous plasmid of pCAMBIA2301 into Isochrysis sp.with the electric shock transformation method,then detected GUS gene of transgenic Isochrysis sp.with the histochemical staining and fluorescence quantitative analysis,and detected GUS reporter gene expression driven by the CaMV35S promoter.The result showed that GUS activity of transgentic Isochrysis sp.was 0.52 nmoL MU/(min·mg).After isolated the total DNA of Isochrysis sp.,the target gene of nptII gene was amplified by PCR,which proved the Plasmid pCAMBIA2301 was integrated into the chromosome of Isochrysis sp..The electricity transformation system of Isochrysis sp.was established in this work,which is the foundation of further development of genetic engineering products.