Beet M14 Strain-specific Expression Of The Gene Encoding Protein Nature

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.