含绿色荧光蛋白及trp3iar基因的草菇表达载体的构建

分别用限制性内切酶EcoR I和HindIII酶切质粒pBGgHg,将切下的含双孢蘑菇gpd启动子、EGFP基因和35S终止子的碱基序列连接到含Ap抗性的pBSK II上,再将这一段序列切下连接到含kan抗性的pCAMBIA1300载体上,形成中间质粒载体YH2873。用限制性内切酶HindIII分别酶切质粒YH2873和质粒pDB06,将从pDB06切下的trp3iar基因连接到中间质粒载体YH2873上,得到表达载体pSAGF。中间载体pYH2873经限制性内切酶EcoR I和HindIII酶切,电泳后显示1.3 kb的目的片断和9 kb的载体片断,表达载体pSAGF经限制性内切酶HindIII酶切,电泳后显示4 kb的trp3iar基因的目的片断和10 kb的载体片断,进一步测序证明重组质粒连接正确,成功构建了草菇的表达载体pSAGF。

Construction of expression vector of Volvariella volvacea with EGFP and trp3iar genes

Plasmid pYH2555 was generated by exciting pBSK II with EcoR I and Hind III and inserting the promoter of gpd,EGFP gene and CaMV 35S terminator sequence from plasmid pBGgHg.Intermediate plasmid pYH2873 was made by digesting pCAMBIA1300 with EcoR I and Hind III and inserting the promoter of gpd,EGFP gene and CaMV 35S terminator sequence from pYH2555.Then pYH2873 was digested by Hind III and ligated to the trp3iar gene which was excited from plasmid pDB06.The plasmid pYH2873 was digested by EcoR I and Hind III, and electrophoresis of the digested products showed two fragments: 1.3 bp fragment and 9 kb fragment.The expression vector pSAGF was digested by Hind III,and electrophoresis of the digested products showed two fragments: 4 kb fragment and 10 kb fragment.Sequencing analysis showed that the recombinant plasmid was correct.Thus the pSAGF,a binary expression vector of V. volvacea,was constructed successfully.