Isolation, Identification Of Nematode-trapping Fungi And The Interactions Of Adhesion Protein From Arthrobotrys Oligospora With Nematode

Animal gastrointestinal nematode is one of the parasitic diseases, which causes hugelosses in animal husbandry annually and impedes the development of livestock. The diseasewas usually controlled by the chemical drugs, however, it is necessary to establish an novelanimal-environment-friendly way to prevent and control the disease for the increasinglydrawbacks of traditional chemical drugs. In recent years, using nematophagous fungi-thenatural enemies of nematodes-to biological to prevent nematodes was received more andmore attentions. As a member of nematode-trapping fungi, with the successful sequencing ofwhole genome and functional studies of important genes of Arthrobotrys oligospora, itprovides a new perspective to elucidate the molecular mechanism of interaction betweenfungal and nematode. The processes of interaction between nematode and fungi can bedivided into attraction, identification, adhesion, colonization and digestion, among whichadhesion is a crucial step, it is the premise of fungi to prey on nematodes. Therefore, thestudy on fungi adhering to nematodes will contribute to understand the molecularmechanisms of interaction between fungi and nematode and to develop anti-nematodesbiological agents.In this study, nematophagous fungi from the Altay region were isolated and identified, andArthrobotrys A1strain was selected as the research subject. A gene encoding adhesion protein(named ADP1) was cloned and then fused with the green fluorescent protein (GFP). Thisfusion protein was expressed in E.coli BL21and purified, and the interaction between ADP1and nematode was confirmed, which provides a base for further study of mechanism offungi-nematodes, and laid a foundation for development of biological agents to controlnematodes diseases. Main research methods and results are as follows:1. Isolation and identification of nematophagous fungi and the induction effect of aminoacids on it trap fomingNematode-trapping fungi were isolated with corn meal agar (CMA) from soil samples ofAltay region in xinjiang province.6strains were successfully isolated that five strainsidentified as Arthrobotrys oligospora and the other one is Arthrobotrys conoides throughmorphological characteristics and molecular biological analysis. In addition, four amino acids(L-phenylalanine, L-tryptophan, L-methionine and L-valine) in different concentrations (0.5,0.05,0.005g/L) were used as revulsive to induce the nematode-trapping organs forming ofAS3strain. The results showed that all the amino acids could effect the traps forming, whilethe L-phenylalanine in concentration of0.05g/L induced maximum number of trapping devices.2. Gene cloning and expression of adhesion molecule ADP1of Arthrobotrys oligosporaA gene encoding adhered protein (ADP1) of A.oligospora (Xinjiang isolates A1) wasamplified by PCR and cloned, and fused with the green fluorescent protein by enzymedigestion method to construct a fusion protein expression vector pET28a-GFP-ADP1. Twovectors (pET28a-GFP-ADP1and pET28a-GFP), respectively, were transformed into E.coliBL21(DE3) for expressing the recombinant protein GFP-ADP1and GFP. The results showedthat the target protein was successfully expressed in E.coli BL21(DE3) after induced by IPTG.SDS-PAGE analysis showed there are two protein bands with the molecular mass of35kDaand50kDa in the supernatant, respectively, which is consistant with expected size of GFP andGFP-ADP1. The expression levels of GFP and GFP-ADP1were approximately8.3mg/mLand6.7mg/mL by UV spectrophotometric detection, respectively.3. Purification of recombinant fusion protein GFP-ADP1and it s interactions withnematodeThe fusion protein GFP-ADP1and GFP were purified, respectively, C.elegans was treatedwith the two recombinant protein. The interaction between fungi and nematode were observedby the fluorescence microscope. The results showed that the green fluorescence can beobserved in all group of GFP-ADP1effect on C.elegans, while no green fluorescence in GFPcontol group could be detected, which indicated that recombinant ADP1can adhere to thenematodes. In addition, this study also found the ADP1could better adhere to nematodes at25and37℃than other temperatures. The adhesion occurred less then10min, which revealedthat ADP1protein played an important role in adhering nematodes.