ABSTRACT
According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.
Subject(s)
Cloning, Molecular , Cytochrome P-450 Enzyme System , Genetics , Metabolism , Eleutherococcus , Genetics , Escherichia coli , Genetics , Metabolism , Gene Expression , Genetic Vectors , Genetics , Metabolism , Plant Proteins , Genetics , Metabolism , Plasmids , Genetics , Metabolism , Recombinant Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To analyze the codon usage of chloroplast genome and the influencing factor in Eleutherococcus senticosus.</p><p><b>METHOD</b>Codon of 52 genes, which were selected from the chloroplast genome sequence of E. senticosus, was multivariate statistical and correspondence analyzed using CodonW and SPSS software.</p><p><b>RESULT</b>GC content at the three position of codons by turns was 46.46%, 38.26%, 29.88%, whereas GC1 and GC2 had a significant correlation coefficient (P < 0.01). The correlation coefficient with GC12, and GC3 was 0.205 and was not significant correlated. There were 30 codons which relative synonymous codon usage was greater than 1 and 29 codons end with A and T. In the corresponding analysis, the first axis shows 10.35% variation. And there was significant correlation coefficient between ENC and GC3. The correlation coefficients with GC3 and ENC were -0.288 and 0.353, respectively. We defined 16 codons from 16 amino acids as the major preference codons in chloroplast genome of E. senticosus.</p><p><b>CONCLUSION</b>The third positions for all codon are preferred to ending with A and T. The codon usage bias is formed under effect of mutation and selection, as well as other factors. But the selection will have a far greater impact than others.</p>