Construction of Setosphaeria turcica STK1-EGFP fusion gene vector and its expression in Pichia pastoris.
- Author:
Yunfeng ZHANG
1
Author Information
- Publication Type:Journal Article
- Keywords: Pichia pastoris; STK1-EGFP fusion gene; Setosphaeria turcica; gene expression
- From: Chinese Journal of Biotechnology 2017;33(6):986-994
- CountryChina
- Language:Chinese
- Abstract: STK1 is one important MAPK gene regulating the conidial development, osmotic stress and pathogenicity of Setosphaeria turcica. At first, the Pichia pastoris GS115 expression vector pPIC3.5K-EGFP containing enhanced green fluorescent protein gene (EGFP) was constructed, then STK1 gene was first amplified by PCR with the template of cDNA of S. turcica model isolate 01-23, and then cloned into the vector pPIC3.5K-EGFP with enhanced green fluorescent protein gene (EGFP) to construct the STK1-EGFP fusion gene expression vector pPIC3.5K-STK1-EGFP. The vector was transformed into the susceptible cells of Pichia pastoris GS115 by electric shock process, and the transformants were identified by MD medium screening and PCR determination. The STK1 gene and EGFP gene could be expressed effectively and stably in the transformants as detected by RT-PCR and fluorescence observation. In addition, we also found that the Kozak sequence before the start codon of STK1 gene could increase 4.8 folds expression level of STK1- EGFP fusion gene. The above research results laid a good foundation for subcellular localization and antibody preparation of STK1 protein.
