玉米大斑病菌STK1与EGFP融合基因载体的构建及其在毕赤酵母中的表达

Construction of Setosphaeria turcica STK1-EGFP fusion gene vector and its expression in Pichia pastoris

摘要:

STK1基因是玉米大斑病菌调控分生孢子发育、渗透胁迫调节和致病性的重要MAPK基因。本文首先构建了含有增强型绿色荧光蛋白基因 (EGFP) 的毕赤酵母GSS115 (Pichia pastoris GS115) 表达载体pPIC3.5K-EGFP，再以玉米大班病菌模式菌株01-23的菌丝cDNA为模板，PCR扩增STK1基因，克隆到pPIC3.5K-EGFP，构建了STK1-EGFP融合基因的GS115表达载体pPIC3.5K-STK1-EGFP。利用电击转化法将该融合基因表达载体转化到GS115感受态细胞内，利用MD培养基筛选、PCR鉴定，获得了STK1-EGFP融合基因的毕赤酵母转化子。通过RT-PCR和荧光观察，发现STK1基因和EGFP基因均可以高效稳定地表达。另外，在试验中我们还发现，在STK1基因起始密码子前加入Kozak序列可以使STK1-EGFP融合基因的表达强度增强4.8倍。以上研究结果为STK1基因表达蛋白的亚细胞功能定位和抗体制备奠定了基础。

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.