Preliminary Functional Analysis Of AMP-Activated Protein Kinase (AMPK) And Protein Kinase A (PKA) In Arthrobotrys Oligospora

Arthrobotrys oligospora is a representative nematode-trapping fungus that can produce three-dimensional networks to prey on nematodes.AMP-activated protein kinase(AMPK)can sense the cellular energy and nutritional status,which can play a role in the regulation of various cellular processes.cAMP dependent protein kinase A(PKA)is an important kinase,which can be activated by cAMP in the cell,and participates in the regulation of multiple biological processes.In this thesis,the biological functions of AMPK and PKA in the mycelia growth and development of A.oligospora were investigated through gene knockout,phenotypic analysis and transcriptome sequencing.Main experimental results:1.The physical and chemical properties,functional domains and cluster analysis of each subunit of AMPK and PKAAMPK contains three subunits,? subunit is the catalytic subunit,and its molecular weight(MW)is 90.57 kDa;? and ? are the regulatory subunits,and their MW are 40.93 and 43.26 kDa,respectively.At the same time,the ? subunit contains more functional domains.The ? and ? subunits of AMPK cluster in different sub-branches of the same evolutionary branch,while the ? subunit clusters in a single branch.PKA also contains three subunits,PkaR is the regulatory subunit,and its MW is 58.47 kDa;PkaC1 and PkaC2 are the catalytic subunits,and their MW are 55.33 kDa and 57.47 kDa,respectively.The PkaR contains a c NMP binding domain,whereas PkaC1 and PkaC2 contain multiple conserved domains or sites,such as AGC kinase,and sites of Ser/Thr kinase and protein kinase ATP binding.The PkaC1 and PkaC2 cluster in the same evolutionary branch,and PkaR clusters separately into one branch.2.Knockout and phenotype comparison of AMPK and PKA subunit coding genesThe coding genes of the three subunits of AMPK were knocked out by homologous recombination technology to obtain knockout strains ?AoAMPK-?,?AoAMPK-? and?AoAMPK-?.Compared with wild-type(WT)strains,the number of conidia and traps of ?AoAMPK-?,?AoAMPK-? and ?AoAMPK-? mutant strains was significantly reduced,and the sensitivity to Congo red was reduced to varying degrees.The sensitivity to menadione is significantly increased.In addition,the utilization of carbon sources except glucose by three mutants was impaired.Similarly,knockout strains ?AoPkaR,?AoPkaC1,and ?AoPkaC2 were obtained by knocking out genes encoding each subunit of PKA.Deletion of AoPkaR and AoPkaC2 caused a reduction in mycelia growth.The conidia yield of ?AoPkaR,?AoPkaC1 and ?AoPkaC2 were all decreased to varying degrees,and the number of traps produced by ?AoPkaC2 after nematode induction was significantly reduced.However,?AoPkaR and ?AoPkaC1 can not produce traps within 48 h induced by nematodes.3.Transcriptome data analysis of ?AoAMPK-?,?AoPkaC1 and WT strains?AoAMPK-? and WT strains were added nematodes to induce 0 h and 12 h,and hyphae samples were respectively collected for transcriptome sequencing.Transcriptome analysis results indicate that AMPK may participate in a series of processes such as energy and nutrient induction,cell wall synthesis,peroxisome regulation,oxidative stress,carbon source utilization,cytoskeleton correlation,and nitrogen source metabolism in A.oligospora.?AoPkaC1 and WT strains were added nematodes to induce 0 h,6 h,12 h and 24 h,and hyphae samples were respectively collected for sequencing.The analysis shows that PKA may be involved in the regulation of biological processes such as transmembrane transport,redox process,mitochondrial function,energy cycle and metabolism in the cell.Innovations of this article:1.This study investigated the functions of AMPK and PKA in A.oligospora.It was found that AMPK is involved in important biological processes such as conidia production,trap formation,resistance to environmental stress and carbon source utilization in A.oligospora.PKA is also involved in the regulation of biological processes such as vegetative growth,conidia formation,trap formation and pathogenicity.2.According to the transcriptome data of ?AoAMPK-?,?AoPkaC1 and WT strains,AMPK and PKA involved in the regulation of various biological processes in A.oligospora.