Cloning and Function Verification of gpd Promoter from Hypoxylon sp.

Abstract

In order to construct the transformation system and study the gene function of Hypoxylon sp.,a highly active promoter element was necessary for the transformation system. In this experiment,the gpd gene and the sequence of about 1000 bp upstream of the gene were cloned by PCR. The results of Neural Network Promoter Prediction,Signal Scan Search Request and PLANTCAREA database analysis showed that the upstream region of the gpd gene contained not only the characteristic core required by the promoter,such as TATA-box,CAAT-box,G-box,GC-box,CT-rich,etc,but also contained a high score transcription start site. To further verify the promoter function of this sequence,the gpd promoter element was ligated with the enhanced green fluorescent protein gene(egfp)and hygromycin gene(hph)to construct a fusion expression vector,which was transformed by Agrobacterium tumefaciens-mediated transformation. Hygromycin resistance screening,green fluorescent protein detection results showed that the gpd promoter of Hypoxylon sp. successfully drove the expression of hygromycin resistance gene and enhanced green fluorescent protein gene,and Agrobacterium tumefaciens-mediated transformation was successful. It laid a foundation for the study of exogenous gene expression and gene function in Hypoxylon sp.