Study On The Functions Of Biosynthesis Related Genes In Arthrobotrys Oligospora Andon Constructions Of Thermomyces Dupontii Mutants

Arthrobotrys oligospo,ra as a model nematode-trapping fungus can produce three-dimensional adhesive traps and kill nematodes.This article pmainly focus on the functions of a cytochrome P450 gene AOL_s00215g21 and 4 biosynthesis related genes AOL_s00043g51,AOL_s00054g811,AOL_s00076g605,and AOL_s00215g535.Bioinformatics analysis revealed 5 candidate genes in the whole genome of A.oligospora:gene AOL_s00215g21 for cytochrome P450,AOL_s00043g51 and AOL_s00076g605 for epigenetic regulators,AOL_s00054g811 for global regulator,AOL_s00215g535 for signal transduction pathway regulator.The knockout vectors for these 4 genes were constructed with the in-fusion method,These 5 genes was disrupted with CaCl2/PEG mediated protoplast transformation.The mutant strains△AOL_s00215g21 and △AOL_s00043g51 showed significantly higher mycelialgrowth,than the wild-type train.In addition,△AOL_s00215g21 displayed increase in spore and trap formations.Compared with the wild-type strain,the growth rates and spore productions of the mutant strains △AOL_s00054g811,△AOL_s00076g605,△AOL_s00215g535 were significantly decreased.The traps production of AOL_s00215g535 was lower than the wild-type strain,but the mortality rate at 24 h was 130%higher than the wild-type strain;at the same time,the HPLC and GC-MS patterns of the three mutant strains showed that the peaks of the metabolic maps were decreased compared with the wild-type strain,but there were still some specific peaks appeared.The HPLC profile of the △AOL_s00215g535 showed that the mutant strain had 8 specific peaks,while lacked 20 peaks which appeared in the wild-type strain;The GC-MS profile of △AOL_s00215g535 showed that the mutant strain had 23 specific peaks,while lacked 5 peaks which appeared in the wild-type strain.Our results suggested that the disruption of these 5 genes led to change of mycelial morphology,growth,spore production,spore germination,trap formation,nematode trapping ability and the synthesis of secondary metabolites of A.oligospora.Among these 5 mutant strains.Mutant △AOL_s00215g535 showed the most obvious difference.In this paper,a preliminary study was carried out on construction of the mutant strains of a thermophilic fungus Thermomyces dupontii 2155.T.dupontii can produce a kind of PKS/NRPS compounds named thermolides,which had a strong inhibitory effect on a variety of pathogenic nematodes.Bioinformatics analysis revealed 9 candidate genes in the whole genome of T.dupontii,including a double-strand break repair enzyme（Ku70）gene Talth₀06364_t1,double-bond reductase（ER）gene Talth₀04946_t1,3 terpene synthases（TPS）genes Talth₀02743_t1,Talth₀03795_t1,Talth₀06604_t1,4 Ⅰ-type polyketide synthases（PKS）genes Talth₀02859_t1,Talth₀04008_t1,Talth₀06479_t1,Talth₀06666_t1.Two mutant strains were obtained by agrobacterium mediated transformation and hygromycin as screening marker:△Talth₀06364_t1 and △Talth₀04946_t1,but the mutants for the disruption of the other 7 genes were not obtained even using nourseothricin as a resistance screening marker and electroporation method.It was speculated that the mechanism of self repair of T.dupontii is too strong,and the conversion method was not accurate enough.In this paper,we preliminarily identified the functions of 5 biosynthetic genes in A.oligospora,the mutant strain △AOL_s00215g535 displayed the most different phenotype compared with wildtype strain.In addition,using agrobacterium tumefaciens transformation and electroporation technology,hygromycin and nourseothricin as resistance screening markers were used for construction of the mutant strain of T.dupontii.