棉花GhPDP基因原核表达条件的优化

Optimization of Prokaryotic Expression Conditions of Cotton GhPDP Gene

丙酮酸脱氢酶磷酸酶GhPDP与棉花雄性不育能量调控方面密切相关。为了得到大量有活性的GhPDP蛋白,试验将对目的蛋白原核表达条件进行优化。将编码GhPDP基因的cDNA序列插入pET-22b中构建重组表达载体pET-22b-GhPDP,转化到大肠杆菌Transetta(DE3)进行IPTG诱导表达,通过单因素试验和正交试验对诱导温度、诱导时间、IPTG终浓度和OD600进行优化,利用SDS-PAGE和GeneTools凝胶分析软件分析目的蛋白的表达量,以摸索目的蛋白可溶性表达的最佳条件。结果显示,成功构建重组表达载体pET-22b-GhPDP;目的蛋白在Transetta(DE3)菌株中表达,表达形式多为包涵体;单因素试验结果表明,在诱导温度20℃、诱导时间为5 h、IPTG终浓度0.10 mmol/L和菌体密度OD600为0.8条件下,目的蛋白可溶性表达量最高;通过正交分析得出,影响目的蛋白可溶性表达的因素强度由高到低依次为诱导温度、IPTG终浓度、诱导时间及菌体密度OD600。综合单因素试验和正交试验结果,确定诱导的最佳条件:菌体密度OD600为0.8、诱导温度为20℃、诱导时间为5 h、IPTG终浓度为0.10 mmol/L。

Pyruvate dehydrogenase phosphatase GhPDP is closely related to the regulation of cotton male sterility energy.To obtain a large number of active GhPDP proteins,the prokaryotic expression conditions the target protein were optimized in this experiment.The cDNA sequence encoding the GhPDP gene was inserted into pET-22b to construct a recombinant expression vector pET-22b-GhPDP,which was transformed into Escherichia coli Transetta(DE3)and induced expression by IPTG.Single factor test and orthogonal experiment were used to optimize the induction temperature,induction time,final IPTG concentration and bacterial cell density OD600,using SDS-PAGE and GeneTools gel analysis software to analyze the expression of the target protein and explore the best conditions for soluble protein expression.The results showed that successfully constructed the recombinant expression vector pET-22b-GhPDP.The target protein was expressed in Transetta(DE3)strain,mainly in the form of inclusion body.The results of single factor experiments showed that the soluble expression level of the target protein was the highest under the conditions of induction temperature of 20℃,induction time of 5 h,IPTG final concentration of 0.10 mmol/L and cell density of OD600 of 0.8.Through orthogonal analysis,it was concluded that the intensity of the factors affecting the soluble expression of the target protein from high to low as induced temperature,final IPTG concentration,induction time,and bacterial cell density OD600.Based on the results of single factor experiment and orthogonal experiment,the optimal induction conditions were determined to be a bacterial cell density of OD600 of 0.8,an induction temperature of 20℃,an induction time of 5 h,and IPTG final concentration of 0.10 mmol/L.