Cloning of gpd-GF Promoter from Grifola frondosa and Constructing of Expression Vector
Two pairs of specific primers were designed according to GPD glyceraldehyde-3-phosphate dehydrogenase gene promoter sequence of Lentinus edodes that has strong promoter activity. Two specific fragments gpd-GF1 and gpd-GF2 were amplified from genomic DNA of Grifola frondosa using primers gpd-GFF1 /gpd-GFR and gpd-GFF2 / gpd-GFR. DNA sequence analysis indicated that gpd-GF1 and gpd-GF2 fragments had 1 018 bp and 615 bp respectively. BLAST search showed that gpd-GF1 and gpd-GF2 had significantly homologous nucleotide sequences with the upstream sequence of gLeGPD gene of Lentinus edodes,the homology was 96 % and 98 % respectively. Promoter Prediction Software analysis showed that fragments gpd-GF1 and gpd-GF2 had strong promoter activity. They contained many important cis-acting elements such as TATA box I box CAAT box GATA box and GCC core. All these elements played important roles in transcription and translation. So gpd-GFF1 might have stronger promoter activity. The above segments were inserted into the expression vector pCAMBIA1301 to displace 35 S promoter that made GUS expression.