Cloning, Expression Of FPD1 Gene In Fusarium Oxysporum F.sp. Cubense And Isolation Of The Biological Control Actinomycete

Banana vasclar wilt, is one of the most widespread, which can seriously affect banana production, caused by Fusarium oxysporum f .sp. cubense (E.F.Smith) snyder et Hansen, Deuteromycotina, Hyphomycetes, Tuberculariales, Fusarium oxysporum.In the studying, Using race 1 and race 4 of Fusarium oxysporum f.sp.cubense as material, analysing pathogenicity-related gene FPD1 of Fusarium oxysporum f. sp. lycopersici, cloning pathogenicity-related gene FPD1 of race 1 and race 4: FPD1 gene of race 1 DNA sequences is 989bp, having an intron of 47 nucleotides, the full length cDNA sequence is 942bp; FPD1 gene of race 4 DNA sequences is 1013bp, having an intron of 47 nucleotides, the full length cDNA sequence is 966bp. Using the software of DNAStar5.0 analysis, race 4 has more length 24 nucleotides than race 1, there are 31 different bases in two nucleotides, 99.3% identity in nucleotides.The FPD1 fragment of race 4, length 615bp, cloned into the pET-32a, finally we got pET-32a-FPD1 expression vector, then transformed pET-32a-FPD1 into expression host BL21(DE3), induced by IPTG, a target band about 46KD can be detected by SDS-PAGE Gel, it indicates that we have expressed FPD1 fragment in E. coli. After SDS-PAGE, the recovery of added the same volume of incomplete Freund Adjuvant emulsified completely, injected rabbits by subcutaneous injection. After fourth injection, blood was harvested by carotid artery and then antiserum was made from it. The titer of antiserum against FPD1 was about 1:10 000 determined by ELISA.A actinomycete was screened using improved Gause's medium, and designated: JFA-001. In the test of antagonistic against race 1 and race 4 of Fusarium oxysporum f.sp.cubense, the result showed this actinomycete could strongly inhibit the growth of race1 and race 4. The sequence of 16S rDNA provides the JFA-001 strain has 99.7% identity in nucleotides.