不同启动子驱动下转基因盐藻外源基因的稳定表达  

Comparison of Stable Expressions of Foreign Genes Driven by Different Promoters in Transgenic Dunaliella salina

作　　者：李杰[1] 曲东京[1] 刘玲玲[1] 冯书营[1] 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物学研究室郑州大学第一附属医院,郑州450052

出　　处：《中国生物工程杂志》2007年第3期47-53,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(30270031)

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,利用编码草丁膦乙酰转移酶的bar基因作为筛选标记,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,经草丁膦(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。结果表明,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。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 tranaformants were isolated from the CaMV-barand 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.

关 键 词：启动子 稳定表达 转基因盐藻 

分 类 号：Q754[生物学—分子生物学]