Controlling The Brontispa Longissima (Gestro) By The MA4 Strain Of Metarhizium Spp. And Cloning And Expressing Its Pr1A Gene Of Cuticle-degrading Proteases And Researched The Antiserum

Brontispa longissima (Gestro) originating from the Asian-Pacific region was very dangerous and devastating for the palm plants. It as an inbreaking insect introduced in our country severely harmed coconut, areca and other palmaceous vegetation, and lead to seriously economical and ecological losing. In this paper a higher virulence strain of Metarhizium anisopliae against the Brontispa longissima (Gestro) was selecting by laboratory detecting virulence. It was named MA4 strain. This paper studied the ITS rDNA region of the MA4 strain, cloned and expressed its Pr1A gene of cuticle-degrading proteases and researched the antiserum of Pr1A recombinant protease, at the same time also studied the relationship between protease and virulence of a higher and a lower virulence strains both in different culture mediums. The results are as follows:(1) Twenty-one different strains of Metarhizium spp. were isolated from soil and cadavers. In the laboratory detected the toxicity of the twenty-one strains by two class screening method. The last result showed that the virulence of the MA4 strain was the most great. It was a better one to control the Brontispa longissium (Gastro).(2) The ITS rDNA region of the MA4 strain were amplified, cloned and sequenced. The whole length of the sequence was 524bp, and was compared with ITS rDNA region of other fungus registered in GenBank. The results showed that the ITS of MA4 strain had 94.2% homologic with Metarhizium anisopliae var. anisopliae. Then the MA4 strain probably was the Metarhizium anisopliae var. anisopliae.(3) The protease production of the higher virulence strain MA4 and the lower virulence strain MA12 was analyzed both in gelatin and in Brontispa longissium (Gastro) cadaver power medium. Both of the two strains the activity of protease in cadaver power medium were higher than that in gelatin, and the MA4 strain was higher than the MA12 strain. What's more, the protease of these two strains was also measured by gelatin and agar plate. The most suitable temperature of producing protease was 28℃both of two strains. There was an obvious linear relationship between protease production and the virulence against Brontispa longissium (Gastro), either in gelatin or in cadaver power medium.(4) The Pr1A gene production has the function of degrading cuticle of insects, was considered one of the key factors to invade insects. The Pr1A gene was amplified by RT-PCR from the MA4 strain. The whole length of this gene was 1242bp, and the nucleotide sequence of the gene was 98% similarity to that of the M. anisopliae accessed in GenBank(M73795). The gene was subcloned into prokaryon expression vector pET-22b(+), transformed this recombinant expression plasmid into E. coli strain BL 21(DE3) and effective expressed. The SDS-PAGE analysis indicated that the recombinant protein was about 42kDa which is same to the reported article. The expression level of recombinant protein was about 63.2% of the whole expressed proteins.(5) After SDS-PAGE, recovered the protein of Pr1A and made antigen, injected rabbits by antigen. After fourth injection, blood was harvested by carotid artery and the antiserum was made from it. ELISA titres was 1/1000, and Western-blotting analysis proved that the recombinant Pr1A protein was reactive to the antiserum. The result indicated that the antiserum was the antibody of the recombinant Pr1A protein.(6) Dot-ELISA with the antibody of the recombinant Pr1A protein to detect the expression of Pr1A in protease production of gelatin and cadaver power medium. Both of that showed positive reaction, but the reaction degree was difference.