灰树花gpd-GF启动子的克隆与表达载体的构建

Cloning of gpd-GF Promoter from Grifola frondosa and Constructing of Expression Vector

根据已经报道有强启动子活性的香菇gpd启动子设计引物,从灰树花基因组PCR扩增获得大小分别为1018,615bp的2个片段gpd-GF1和gpd-GF2。通过DNA序列测定得知二者的大小分别为1018,615bp。经NCBI中的BLASTN比较,gpd-GF1,gpd-GF2与已报道的香菇中克隆的gLeGPD基因上游序列的同源性分别为96%,98%。通过启动子预测软件分析,结果表明,gpd-GF1和gpd-GF2含有多个顺式作用元件如TATAbox,GATAbox,CAATbox等,初步证实gpd-GF1,gpd-GF2可能有较强的启动子活性。将gpd-GF1,gpd-GF2分别与切除CaMV35S启动子的pCAMBIA1301大片段进行亚克隆,构建成表达载体pCBgpdGF1和pCBgpdGF2。

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-GF 1 and gpd-GF2 were amplified from genomic DNA of Grifolafrondosa using primers gpd-GFF 1 /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 pCAMBIA 1301 to displace 35 S promoter that made GUS expression.