甜菜M14品系特异表达基因编码蛋白性质的研究
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摘要
甜菜M14品系具有无融合生殖特性。为了研究甜菜M14品系特异表达基因M14-263和M14-341编码蛋白的性质,为进一步将两基因转化入模式植物奠定基础,解开基因M14-263和M14-341的功能之谜,本实验以pET-28a为基础载体,利用E.coli BL21(DE3)表达体系,将甜菜M14品系表达基因M14-263和M14-341的cDNA全长分别重组于原核表达载体pET-28a,成功建立了甜菜M14品系特异表达基因M14-263和M14-341的原核表达体系。经SDS-PAGE凝胶电泳及Western印迹检测表明,两个融合蛋白正确表达,其中M14-263融合蛋白的分子量约为26kD,M14-341融合蛋白分子量约为36kD。
为了优化融合蛋白表达条件,提高蛋白表达量,本实验选择可能影响蛋白表达的四个因素(培养时间、IPTG浓度、诱导时间、诱导温度)作为研究对象,设计了四因素三水平正交实验。极差和方差分析结果显示:影响融合蛋白M14-263表达条件的关键因子为诱导时间,培养时间和诱导温度对M14-263蛋白表达量有一定的影响作用,而IPTG浓度的影响效果不显著;影响融合蛋白M14-341表达条件的关键因子为诱导温度,诱导时间和IPTG浓度的影响次之,培养时间的影响不显著。实验结果表明,融合蛋白M14-263的最优表达条件为:IPTG浓度1.2mmol/L,培养时间3h,诱导时间6h,诱导温度37℃,此条件下获得的融合蛋白M14-263占菌体总蛋白的百分比高达33.2%,融合蛋白M14-263浓度为401.9mg/L;融合蛋白M14-341的最优表达条件为:IPTG浓度0.8mmol/L,培养时间3h,诱导时间7h,诱导温度28℃,此条件下获得的融合蛋白M14-341占菌体总蛋白的百分比较优化前显著提高,达16.1%,融合蛋白M14-341浓度为70.98mg/L。
热稳定性研究结果表明,M14-263融合蛋白和M14-341融合蛋白均较稳定。亲水性研究结果表明,M14-263融合蛋白较亲水,与前期生物信息学预测结果一致,但M14-341融合蛋白在前期生物信息学预测中呈疏水性,在本实验中则表现出很强的亲水性,可能是由于蛋白折叠后含有多个亲水的α螺旋,或者由于附加序列的作用导致其具有亲水性。
M14 is a monosomic addition line with the characteristic of apomixis. To research the property of M14-263 protein and M14-341 protein expressed by M14-263 and M14-341 of specially expressed genes of M14,and lay the foundation of the study about the transformation of of M14-263 gene and M14-341 gene into model plants,the two gene were recombinated to the expression region of prokaryotic vector pET-28a respectively,as a basic vector,resulted in the successful construction of prokaryotic expression system of M14-263 gene and M14-341 gene by E.coli BL21(DE3)expression system. By the SDS-PAGE and western blotting,the results showed that two fusion proteins could be expressed correctly,the molecular weight of M14-263 fusion protein is about 26kD,and the molecular weight of M14-341 fusion protein is about 36kD.
To optimize the expression conditions of M14-263 gene and M14-341 gene,raise the production of the two fusion protein,four effect factors were chose for a four-factored,three -leveled orthogonal experiment. The results obtained from range analysis and variance analysis showed that the key factor in the expression conditions of M14-263 is the inducement time, the effect of the inducement temperature and the culture time are weaker, and the concentration of IPTG had no significant influence. The key factor in the expression conditions of M14-341 is the inducement temperature,the inducement time and the concentration of IPTG had some effect,and the culture time had no significant influence. By validating experiments,the optimum expression condition of M14-263 gene is that the concentration of IPTG is 1.2mmol/L,the culture time is 3h,inducement time is 6h,and inducement temperature is 37℃. In this condition,the percentage of M14-263 fusion protein in total proteins is up to 33.2%; the concentration of M14-263 fusion protein is 401.9 mg/L. The optimum expression condition of M14-341 gene is that the concentration of IPTG is 0.8mmol/L,the culture time is 3h,inducement time is 7h,and inducement temperature is 28℃. In this condition,the percentage of M14-341 fusion protein in total proteins reaches 16.1%; the concentration of M14-341 fusion protein is 70.98 mg/L.
The two fusion proteins were both stable in the experiment of thermal stability. In the experiment of hydrophilicity,M14-263 fusion protein showed hydrophilicity,which is in accordance with the previous bioinformatics analysis results,and M14-341 fusion protein also showed hydrophilicity,which is different from the theory analysis results. For this difference,two aspects can be considered. Firstly,M14-341 fusion protein forms many hydrophilicα-helix structures from the previous bioinformatics analysis results in the hydrophilicity. Secondly,foreign sequence from vector can influence the property of M14-341 fusion protein.
为了优化融合蛋白表达条件,提高蛋白表达量,本实验选择可能影响蛋白表达的四个因素(培养时间、IPTG浓度、诱导时间、诱导温度)作为研究对象,设计了四因素三水平正交实验。极差和方差分析结果显示:影响融合蛋白M14-263表达条件的关键因子为诱导时间,培养时间和诱导温度对M14-263蛋白表达量有一定的影响作用,而IPTG浓度的影响效果不显著;影响融合蛋白M14-341表达条件的关键因子为诱导温度,诱导时间和IPTG浓度的影响次之,培养时间的影响不显著。实验结果表明,融合蛋白M14-263的最优表达条件为:IPTG浓度1.2mmol/L,培养时间3h,诱导时间6h,诱导温度37℃,此条件下获得的融合蛋白M14-263占菌体总蛋白的百分比高达33.2%,融合蛋白M14-263浓度为401.9mg/L;融合蛋白M14-341的最优表达条件为:IPTG浓度0.8mmol/L,培养时间3h,诱导时间7h,诱导温度28℃,此条件下获得的融合蛋白M14-341占菌体总蛋白的百分比较优化前显著提高,达16.1%,融合蛋白M14-341浓度为70.98mg/L。
热稳定性研究结果表明,M14-263融合蛋白和M14-341融合蛋白均较稳定。亲水性研究结果表明,M14-263融合蛋白较亲水,与前期生物信息学预测结果一致,但M14-341融合蛋白在前期生物信息学预测中呈疏水性,在本实验中则表现出很强的亲水性,可能是由于蛋白折叠后含有多个亲水的α螺旋,或者由于附加序列的作用导致其具有亲水性。
M14 is a monosomic addition line with the characteristic of apomixis. To research the property of M14-263 protein and M14-341 protein expressed by M14-263 and M14-341 of specially expressed genes of M14,and lay the foundation of the study about the transformation of of M14-263 gene and M14-341 gene into model plants,the two gene were recombinated to the expression region of prokaryotic vector pET-28a respectively,as a basic vector,resulted in the successful construction of prokaryotic expression system of M14-263 gene and M14-341 gene by E.coli BL21(DE3)expression system. By the SDS-PAGE and western blotting,the results showed that two fusion proteins could be expressed correctly,the molecular weight of M14-263 fusion protein is about 26kD,and the molecular weight of M14-341 fusion protein is about 36kD.
To optimize the expression conditions of M14-263 gene and M14-341 gene,raise the production of the two fusion protein,four effect factors were chose for a four-factored,three -leveled orthogonal experiment. The results obtained from range analysis and variance analysis showed that the key factor in the expression conditions of M14-263 is the inducement time, the effect of the inducement temperature and the culture time are weaker, and the concentration of IPTG had no significant influence. The key factor in the expression conditions of M14-341 is the inducement temperature,the inducement time and the concentration of IPTG had some effect,and the culture time had no significant influence. By validating experiments,the optimum expression condition of M14-263 gene is that the concentration of IPTG is 1.2mmol/L,the culture time is 3h,inducement time is 6h,and inducement temperature is 37℃. In this condition,the percentage of M14-263 fusion protein in total proteins is up to 33.2%; the concentration of M14-263 fusion protein is 401.9 mg/L. The optimum expression condition of M14-341 gene is that the concentration of IPTG is 0.8mmol/L,the culture time is 3h,inducement time is 7h,and inducement temperature is 28℃. In this condition,the percentage of M14-341 fusion protein in total proteins reaches 16.1%; the concentration of M14-341 fusion protein is 70.98 mg/L.
The two fusion proteins were both stable in the experiment of thermal stability. In the experiment of hydrophilicity,M14-263 fusion protein showed hydrophilicity,which is in accordance with the previous bioinformatics analysis results,and M14-341 fusion protein also showed hydrophilicity,which is different from the theory analysis results. For this difference,two aspects can be considered. Firstly,M14-341 fusion protein forms many hydrophilicα-helix structures from the previous bioinformatics analysis results in the hydrophilicity. Secondly,foreign sequence from vector can influence the property of M14-341 fusion protein.
引文
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