黄肿树JcFATA基因原核表达载体构建及表达体系优化

Construction of Prokaryotic Expression Vector of Jc FATA Gene and Optimization of Expression System

植物脂酰-酰基载体蛋白硫酯酶A(fatty acyl acyl carrier protien thioesterase,FATA)是脂肪酸积累过程中的关键酶,直接调控脂肪酸的含量与组成。目前有关黄肿树脂酰-ACP硫酯酶A基因(Jc FATA)(Gen Bank登陆号:EU267122.2)的功能研究还未见报道。对Jc FATA基因进行原核表达是研究Jc FATA基因功能的一种重要手段,而开展这项研究的前提是构建原核表达载体。因此,本研究以黄肿树c DNA为模板克隆出Jc FATA基因原核表达片段,将其连接到带有His标签的原核表达载体p Cold I上,将所构建的重组载体p Cold I-Jc FATA转入大肠杆菌Rosseta。为了表达更多的可溶性His-Jc FATA融合蛋白,本文还探讨了不同IPTG浓度(0.1、0.5、1.0 mmol/L)、不同诱导温度(15、25、37℃)和不同诱导时间(0、15、30 h)对蛋白表达的影响,诱导产物经超声波裂解破碎后,利用SDS-PAGE比较分析融合蛋白表达量,从而确定最佳的表达体系。结果表明,当添加诱导剂IPTG的浓度为0.5 mmol/L,诱导时间为15 h,培养温度为25℃时,可以获得较多的可溶性融合蛋白(His-Jc FATA)。此外,进行蛋白纯化时发现,在镍柱上进行第5次洗脱时所获得纯化蛋白质量最佳。本研究的结果可为进一步研究Jc FATA基因的功能奠定基础。

The fatty acyl carrier protien thioesterase（FATA） is a key enzyme in the accumulation of fatty acids,which directly regulates the content and composition of fatty acids.The function of the acyl-ACP thioesterase A gene（Gen Bank accession number：EU267122.2） in Jatropha curcas has not been reported before.An important method to analyze the function of JcFATA gene is the prokaryotic expression,and the prerequisite of which is to construct prokaryotic expression vector.The JcFATA gene was cloned from J.curcas c DNA and inserted into the prokaryotic expression vector p Cold I with His-tag.The recombinant vector p Cold I-JcFATA was transformed into Escherichia coli Rosseta.In order to express more soluble fusion proteins His-JcFATA,different IPTG concentrations（0.1,0.5 and 1.0 mmol/L）,different induction temperatures（15,25 and 37 ℃） and different induction times（0,15 and 30 h） were investigated on the protein expression.After the cells were disrupted by ultrasound,the expression of the fusion protein was analyzed by 12% SDS-PAGE to determine the optimal expression condition.The results showed that the soluble fusion protein（His-JcFATA） could be obtained better when the concentration of the inducer IPTG was 0.5 mmol/L,the induction time was 15 h,and the culture temperature was 25 ℃.In addition,the study also showed that the fifth elution for protein purification on the nickel column might obtain the best quality of purified protein.The results of this study can lay a foundation for the further analyzing functions of JcFATA gene.