Radish Antifungal Protein Rs-afp <sub> 2 </ Sub> Gene Pcr Site-directed Mutagenesis And Expression In E. Coli

AbstractRaphnus sativus -antifun gal protein2 (Rs-AFP,) was isolated from the seeds of radish. It was a small , cysteine-rich, defense-related antifungal protein. The encoding sequence was 243 bp. It encoded 80 amino acids which including signal peptide of 29 amino acids and mature peptide of 51 amino acids.Seven primers had been synthesized according to the Rs-AFP2 gene sequences and preference of the genetic code in the gene expressions and regulations. The splicing by overlap extension was applied to change the bare codon AGG into favored codon CGA, which encoded the ninth amino acid in the gene encoding Rs-AFP2. Some other bases were also mutated in the beginnig of the encoding parts. The mutated gene was quickly cloned by nested PCR. PCR product of Rs-AFP2 gene was retrieved by low melting agaroses(1 .5% wt/vol) gel electrophoresis, then was inserted into pGEM-5Zf/EcoRV-T easy vector. The recombination plasmid was named pOEM-Tm and was identified by digestions with EcoR I, PCR and sequence analysis, thereby proving that the gene alteration had been completed successfully.In order to remove the signal peptide sequences of Rs-AFP2 eDNA, two different upstream primers, both of which had a 5'-terminal Nde I site, were designed and synthesized. Each could match with sequences either Rs-AFP2 or Rs-AFPm. The downstream primmer had a 5'-terminal Xho I site. The coding sequences of the mature peptides were amplified by using the respective recombinant cloning vectors as templates. Then the digested products were inserted into pET2lb vectors at the site of Nde I and Xho I to construct expression vectors pETAFP,~ffl pETAFPm, which had not the signal peptide at all.The two expression vectors, which were constructed by the way of the maturepeptides, were expressed in E. coli BL2 1 strain, and the products were identified by SDS-Ticine-PAGE. The inhabition experimelfls showed that the inhibition activity of pETAFPm expression product was much stronger than that of pETAFP2 one. Therefore the mutated gene had improved the expression efficiency validly as a result.