棘孢木霉Myb27转录因子基因特性分析、原核表达及产物纯化

Characteristics Analysis,Prokaryotic Expression and Product Purification of Trichoderma asperellum Myb27 Transcription Factor

以棘孢木霉CGMCC11653菌株菌丝体总RNA反转录的cDNA为模板,克隆全长717 bp转录因子Myb27基因编码序列,其编码238氨基酸组成的多肽。用生物信息学软件预测蛋白的理化性质、结构域并进行亚细胞定位、氨基酸多序列比对和进化关系分析,结果表明:其相对分子质量为27000,属于Myb-like DNAbinding结构域的蛋白,亚细胞定位在细胞核中。链格孢菌毒素诱导条件下Myb27的表达有显著变化,可能参与棘孢木霉抗链格孢菌毒素胁迫应答反应的正向调控。构建原核表达载体pMAL-Emyb27,转化大肠杆菌ER2523获得转化子ER2523-Emyb27,其成功诱导的最适条件为0.1 mmol/L IPTG 37℃连续诱导培养3 h;用PBS+10 mmol/L麦芽糖洗脱层析柱可得到与MBP标签融合的单一可溶性69500重组rMyb27蛋白。可见Myb27转录因子调控抗病基因的表达所结合的顺式作用元件提供体外重组蛋白,为调控棘孢木霉抗杨树叶枯病链格孢菌毒素胁迫的机理提供理论依据。

Using cDNA of the total RNA reverse transcription from the mycelium of Trichoderma CGMCC11653 strain as a template,the full-length 717 bp transcription factor Myb27 coding sequence was cloned,encoding 238 amino acids polypeptide.The bioinformatics software was used to predict the physical and chemical properties,structural domains and protein subcellular localization,and to analyze the multi-sequence alignment of amino acids and the evolutionary relationship.The results showed that the protein belonged to Myb-like DNA-binding domain and the subcell was located in the nucleus with a molecular weight of 27000.The expression of Myb27 changed significantly under the induction of Alternaria toxin,which may be involved in the positive regulation of the response of Trichoderma against Alternaria toxin stress.The prokaryotic expression vector,pMALEmyb27,was constructed,and the transformant ER2523-Emyb27 was obtained by transforming E.coli ER2523.The optimal conditions for successful induction of ER2523-Emyb27 were 0.1 mmol/L IPTG 37℃ for 3 h.A single soluble 69500 recombinant rMyb27 protein was obtained by eluting the MBP label with PBS+10 mmol/L maltose chromatography column.This study has provided recombinant proteins in vitro for the cis-acting elements bound by Myb27 transcription factor to regulate the expression of disease resistance genes,and theoretical basis for the mechanism of regulating Trichoderma against toxin stress of Alternaria alternata.