Functional Alanlysis Of Ifcdp1 Gene From Isaria Fumosorosea

Isaria fumosorosea is a promising insect biological control agent widely used in pest management.The full-length I.fumosorosea cuticle-degrading protease gene,Ifcdp1,genomic sequence was cloned by TALL-PCR using DN A walking methods.The Ifcdp1gene disruption strain?mutant?and complemented strain resulted by disruption Ifcdp1 gene from the wild type strain and supplement mutant strain with Ifcdp1 gene via Agrobacterium-mediated transformation?AMT?.The function of Ifcdp1 gene was evaluated on base of comparing the biological characteristics,biochemical characteristics of metabolite and pathogenicity of WT and MT strains.The research was presented as below:?1?Amplification and analysis of the full-length of Ifcdp1 genomic sequence.The full-length of Ifcdp1 genomic sequence was cloned by TALL-PCR using DNA walking methods.The complete nucleotide sequence of Ifcdp1has been deposited in Genbank?accession number KU519628?.Analysis of the Ifcdp1open reading frame?ORF?with the length of 1140 bp indicated the present of three introns?65 bp,59 bp and 59 bp in length,respectively?.A potential TATA box was identified by online prediction at position-508 by upstream of the start codon.Southern blot analysis showed the presence of a single Ifcdp1gene in wild type strain.The deduced translated protein product of the Ifcdp1 gene encoded a polypeptide of 380 amino acid residues,with a calculated molecular mass of 39.3 k Da and a Isoelectric point of 8.72.Alignment of the conserved glycosyl hydrolase peptidases S8-S53 superfamily region including the active site of Asp₁₄,His₁₇₁ and Ser₃₂₃,was found in this deduced translated protein of theIfcdp1.?2?Generation of Ifcdp1 disruption and complemented stains.The Ifcdp1 gene knockout and complementation vectors were constructed and yielding three plasmids including pPB-5'-flanking,p BT-3'-flanking and p Ben-Ifcdp1,respectively.The two plasmids pPB-5'-flanking and p BT-3'-flanking were transformed into wild type strain via agrobacterium mediate transformation?AMT?yielding disruption strain?mutant?,and the plasmid p Ben-Ifcdp1 were transformed into mutant strain via AMT yielding complemented strain,respectively.New transformants were single spore purified and the correct integration events verified by PCR.?3?Effect of Ifcdp1 on biological characteristics of Isaria fumosorosea.Disruption of the Ifcdp1 gene in I.fumosorosea resulted in no effects on vegetative growth rates.The mycelium growth rates among the three strains,including mutant strain?MT?,wild type strain?WT?and the complemented strain?C T?,were not significantly different when grown at the same temperature and on the same medium,respectively.Loss of the Ifcdp1 gene resulted in significant affects on co nidial production.Overall conidial production in the MT was significantly lower,almost half,than that of the WT when grown on the same medium.No significant difference in total protease activity was seen between WT,MT and CT strain grown in CZB medium.But the Pr1 activity of MT grown in CZB,PDB and SDBY decreased sharply during the growth phase 24-48h as compared to that of WT.?4?Effect of knock outIfcdp1gene of I.fumosorosea on insect pest mortality.Insect bioassay using the whitefly,Bemisia tabaci,nymphs showed decreased infectivity for the MT,i.e.lethal dose(LC₅₀)of MT?2.26×10⁶conidia/ml?higher than that of WT?5.11×10⁵conidia/ml?,and an increased time to death(LT₅₀)from 130?WT?to 176 h?MT?.In contrast,insect bioassay using the diamondback month,Plutella xylostella,larvae revealed increased infectivity,decreased in LC₅₀?1.03×10⁶conidia/ml for WT/1.59×10⁵conidia/mL for MT?,and a decreased LT₅₀ from 78?WT?to 62 h?MT?.Infection by I.fumosorosea MT or WT resulted in sharp decrease in the total number of egg production.The fecundity of B.tabaci infected by MT was significantly higher than that of whitefly infected by WT.