不同启动子驱动下转基因盐藻外源基因的稳定表达

摘要：为了探讨外源性与内源性启动子对转基因盐藻外源基因表达的影响，将含外源性启动子CMV35S的表达载体CMV35S-bar（G12）和含内源双拷贝碳酸酐酶启动子DCA1的表达载体DCA1-bar（D-B）分别转化盐藻，筛选稳定转化株后，观察在不同启动子驱动下外源基因的表达情况及对转基因盐藻生长的影响。 通过电击法分别将表达载体G12 和D-B转化盐藻，经PPT筛选后，各得到了3株PPT抗性藻株，经PCR及测序分析证实外源基因bar已经整合到盐藻的基因组中，半定量RT-PCR结果显示，在内源性启动子DCA1驱动下，bar基因的表达强度明显高于在外源性启动子驱动下bar的表达，并且D-B转化株的bar基因表达在盐诱导下其表达明显提高，而G12转化株中bar基因的表达对盐诱导无反应。Southern blot 分析显示，外源基因的拷贝数与不同启动子间无相关性。转化株的生长特性分析显示，D-B转化株的生长速度明显高于G12转化株。本研究的结果指出，内源诱导型启动子在驱动转基因盐藻外源基因的高效稳定表达中比外源组成型启动子更具有优势。

Comparison of stable expressions of foreign genes driven by different promoters in transgenic Dunaliella salina

The purpose was to compare the difference between transgene expressions driven by homologous duplicated carbonic anhydrase (DCA) promoter and foreign CaMV35S promoter in the unicellular green alga, Dunaliella salina(D.salina).The CaMV35S promoter-bar construct and DCA promoter-bar construct into D.salina by a Backon 2000 electroporation system were introduced. After the repeated selections with the phosphinothricin (PPT) of 3mg/L, 3 PPT-resistant phenotype transformants were isolated from the CaMV-bar and DCA-bar pools of transformants of D. salina, respectively. The results of PCR and sequencing showed that bar genes were stably integrated into the genome of D.salina, and Southern bolts showed the number of transgene copy had no significant difference between both promoters. Semi-quantitive RT-PCR indicated that the mRNA levels of bar gene were higher in DCA-bar transformants than the CaMV-bar transformants, and could be increased under the induction of high salt in DCA-bar transformants but not in the CaMV-bar transformants. Analysis of growth rate of transformants showed DCA-bar transformants achieved the log stage faster than the CaMV-bar transformants. It is concluded that the homologous promoters have more advantages than the foreign promoters in the transgenic D.salina.