香灰菌<i>gpd</i>启动子的克隆及功能验证

陈悦; 刘冬梅; 许丹云; 王媛媛; 马爱民

doi:10.13386/j.issn1002-0306.2019.17.016

香灰菌gpd启动子的克隆及功能验证

Cloning and Function Verification of gpd Promoter from Hypoxylon sp.

摘要

摘要: 为了构建香灰菌的转化体系,研究香灰菌的基因功能,需要获得高活性的启动子元件。本实验通过PCR技术分别克隆得到gpd基因以及其基因上游约1000 bp序列,使用Neural Network Promoter Prediction、Signal Scan Search Request、PLANTCAREA database软件分析发现,gpd基因上游区域内不仅包含启动子所需的特征核心元件TATA-box、CAAT-box、G-box、GC-box、CT-rich等,还含有一个高分值的转录起始位点。为进一步验证该序列的启动子功能,将gpd启动子元件与增强型绿色荧光蛋白基因(egfp)和潮霉素基因(hph)连接构建成融合表达载体,通过根癌农杆菌介导将其转化香灰菌丝。潮霉素抗性筛选、绿色荧光蛋白检测结果显示,香灰菌gpd启动子成功驱动了潮霉素抗性基因和增强型绿色荧光蛋白基因的表达,根癌农杆菌介导转化成功,为香灰菌外源基因表达及基因功能的研究奠定了基础。

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.