Isolation And Purification Of An Antifungal Protein Against American Ginseng Pathogens And Synthesis Of Its Gene And Expression In E.coli

American ginseng (Panax quinquefolium L.), a valuable Chinese herb, was introduced from USA and Canada and cultivated in China and was well received by the people all over the world for its active ingredients as immunostimulant. Severe fungus diseases often occurred in the process of growth and resulted in an enormous reduction of American ginseng output and up to now there is no effective way to control them. Antifungal proteins and the application of their genes can be a novel and effective way. Through the preliminary selection of antifungal activity of crude extracts from 12 plants, strong antifungal activity was found in pokeweed seeds. A peptide with 7,000Da was isolated and purified by aqueous extraction, heating process, ion exchange column chromatograph(CM-22), and Gel filtration(Sephadex G-25) from pokeweed seed. It is very basic peptide with its PI about 11 and the amino acid sequence of N-terminal is Ala-Gly-Gys-Ile-Lys-Asn-Gly. By comparison of amino acid sequence and molecular weight, it is verified that the isolated peptide is PAFP-S. The peptide showed obvious inhibitory activity against the growth of 3 main pathogens of American ginseng: Rhizoctonia solani, Altemaria panax, Fusarium sp., while no action on the growth of Phytophthora cactorum.It suggested that PAFP-S might be a chitin-binding protein. According to the amino acid sequence of PAFP-S, four oligonucleotides primer1 (65bp), primer2(65bp), primer3(65bp),primer4(64bp) which cover the full peptide gene were chemically synthesized using E.coli-preferred codons. The gene of PAFP-S was amplified by recursive PCR and was cloned to pGEM-T vector and a recombinant pGEMT-210 was formed. Sequence analysis of pGEMT-210 showed that the cloned gene was identical with that of the designed. This is the first synthesized gene for PAFP-S and there has no cloned gene for pafp-s.A nonfusion prokaryotic expression vector pRSETB-210 and a fusion prokaryotic expression vector pTrcA210 for PAFP-S gene were constructed. The result of tricine-SDS-PAGE revealed the nonfusion protein of pafp-s gene was expressed at 0.67% with the introducton of IPTG and sequence analysis of the nonfusion expressed protein show that N-terminal 15 amino acid sequence was the same as that of native PAFP-S, which further demonstrated the fusion protein of pafp-s gene was expressed. On the expression vector pTrcA210, a fusion protein of pafp-s gene was expressed by 2.26% with induction of IPTG.The synthesized antifungal gene of pafp-s has great potentials in application, especially in the disease-resistance breeding of crops.