香菇gpd启动子引导的云芝漆酶基因真核表达载体的构建

Construction of Eukaryotic Expression Vector of Laccase Gene (lcc1) from Coriolus versicolor under the Control of Lentinus edodes GPD Promoter

根据Genbank中提交的云芝(Coriolus versicolor(L.)Quel.)漆酶(lcc1)基因cDNA序列设计引物,利用RT-PCR获得云芝漆酶基因cDNA完整编码框,与GenBank中云芝漆酶基因同源性为96%;以pCAM-BIA1381xb为初始载体,利用引物两端的酶切位点,将克隆得到的云芝漆酶基因cDNA序列分别连接到香菇gpd启动子Lg1和Lg2的下游,构建成香菇启动子引导的云芝漆酶基因真核表达载体pC-XG1-YZ-Lac和pC-XG2-YZ-Lac,利用冻融法将其转到了农杆菌EHA105中。

A pair of primers was designed according to the reported cDNA sequence of laccase gene, and the total RNA was extracted from mycelium of Coriolus versicolor. An amplification product about 1 563 bp was obtained by RT-PCR reaction. Sequence analysis showed that the cloned eDNA shared a 96 percent homology in comparison with the reported data. Two express vectors, pC-XG1 -YZ-Lac and pC-XG2-YZ-Lac, were constructed by restriction enzyme digestion and legation using pCAMBIA1 381 xb as initial vector, and in these two vectors, the laccase eDNA of C. versicolor was under the control of two L. edodes GPD promoters Lgl and Lg2, respectively. These two vectors were transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method.