The Disruption,Complementary And Function Analysis Of CAP(adenylate Cyclase-Associated) Protein In Thermomyces Lanuginosus

Thermomyces lanuginosus is a thermophilic fungus that is closely related to the daily life of mankind and has the highest growth temperature in eukaryotes.In previous studies,a variety of thermophilic enzymes have been isolated from this fungus.Expressed in eukaryotes,they have been widely used in industrial,agricultural and microbial research,which have great influence on medicine,brewing,fermentation,waste treatment and other fields.In recent years,the researchers have focused on the cloning of thermophilic protease,lipase,glucoamylase,chitinase and xylanase,and its physiological and biochemical properties.The genetic function of specific gene has not been reported in this fungus so far.Magnaporthe oryzae as a kind of filamentous fungi,the loss of CAP affects the cell viability and the appressoria formation and then reduces the pathogenicity.The CAP homology gene was also found in the Thermomyces lanuginosus by sequence alignment,and the homology with the CAP1 gene in the rice blast fungus was 42.75%.By bioinformatics analysis,CAP is a member of the CAP_C superfamily,which contains the CARP domains,highly conserved.The subcellular localization of CAP is on the cell membrane.NJ method to establish phylogenetic tree and analysis of genetic relationship shows that: Aspergillus lentulus evolution of the recent distance,and Homo sapiens,Mus musculus evolution distance farthest.In order to further explore the function of CAP,we constructed the knockout vector of CAP gene and the complementary/subcellular localization vector,and then used ATMT method to transform the conidia of Thermomyces lanuginosus to obtain CAP knockout mutant ?CAP,complementary/subcellular mutant ?CAP/CAP was obtained by PEG-mediated genetic transformation.With wild-type strain 9w-WT,knockout strain ?CAP,CAP complementary strains CH07570-?CAP,CH14404-?CAP,MGG01722.6-?CAP as the research materials,results showed that: Compared with 9w-WT,the colony growth rate of ?CAP is slightly faster;the aerial hyphae of ?CAP is similar to 9w-WT.The spores of the above-mentioned strains were observed.The result showed that the spore of 9w-WT were smooth and hollow,but the ?CAP was spiked and irregular spheroids,CH07570-?CAP,CH14404-?CAP showed hollow spherical spheres with large differences in size,and the complementary strain MGG01722.6-?CAP were irregular spherical sphere with surface roughness.This biological feature maybe related to CAP involvement in cell surface recognition.Therefore,CAP is essential to maintain the vegetative growth of Thermomyces lanuginosus.The spore germination rate of ?CAP was not significant difference compared with the 9w-WT,and the spore germination rate of the complementary strains was similar to that of the wild-type strains.Thus,CAP does not affect the spore germination activity of Thermomyces lanuginosus.The relevant stress experiments were peformed,1.H2O2 stress experiment: compared with the 9w-WT,the H2O2 sensitivity of ? CAP decreased,but the difference was not significant(no data available).Similar to the colony morphology of the strains on PDA medium,colony color change,corresponding to the sporulation of strains.2.Cell wall integrity test: compared with 9w-WT and complementary strain,?CAP in Congo-Red and SDS CM medium are not sensitive,thus CAP is concerned with the integrity of the cell wall in Thermomyces lanuginosus,and the expression of CAP is response to basic adversity from external environment.3.Osmotic stress experiment: compared with the 9w-WT,the knockout strain was more sensitive to the osmotic pressure factor and the growth of Thermomyces lanuginosus is inhibited,and the complementary strain recovered the osmotic stress resistance of ?CAP partially.In summary,CAP is essential to maintain the growth and development of Thermomyces lanuginosus and stress response.To investigate the effect of exogenous c AMP on knockout and complementary strains,c AMP was added to CM and PDA medium.The results showed that colony color were brown and penetrated into the medium,which may be related to the metabolites production and sporulation;we also found that exogenous c AMP could effectively compensate for CAP deficiency on growth and development of Thermomyces lanuginosus.Besides,the sporulation could be partially or fully restored.Above all,a pathogenicity-related CAP1 gene in Magnaporthe oryzae was used as a starting point,and it was found that there is CAP in Thermomyces lanuginosus by sequence contrast.Through a series of bioinformatics analysis,knockout and complement/subcellular localization strains were obtained by the basic experiment,and then the function of CAP was studied.Which provide some theoretical basis and method guidance for the study of specific gene of Thermomyces lanuginosus or other thermophilic fungi.It also open a new perspective on the genetic function of thermophilic fungi related genes,meanwhile,it be able to ascertain thermophilic mechanism of thermophilic fungi as soon as possible.