Preliminary Study On The Functions Of MAPK And RGS4 Proteins Of Myxospora Spores

Dactylellina haptotyla is a kind of nematode-trapping fungi(NTF),whose trap is a unicellular spherical structure called the knob.Meerupati et al.(2013)have recently sequenced the genome of D.haptot D.and detected many similarities to the genome of thenet-forming nematode-trapping fungus Arthrobotrys oligospora.G protein is a ubiquitous signal regulatory protein in the fungus,and the G protein signaling factor(RGS)activates the GTPase activity of the Ga subunit,thereby regulating the signaling pathway downstream of the G protein.MAPK is a key component of the downstream signaling pathway of G protein.The functions of RGS and MAPK have been reported in some filamentous fungi,but their functions have not been reported innematode-trapping fungi.In this study,the functions of Mapk and Rgs4 genes in D.haptotyla were analyzed during the growth and development using the gene knockout technology.The main results:1.Functional domains of MAPK and RGS4 proteins in D.haptotyla and cluster analysis.MAPK protein contains 415 amino acids with a predicted molecular weight of 47.1 kDa and an isoelectric point of 5.41.The protein contains a conserved domain protein kinase-like domain(IPR011009)and multiple conserved functional sites,such as protein kinase,ATP binding site(IPR017441).The RGS4 protein contains 1278 amino acids with a predicted molecular weight of 142.9 kDa and an isoelectric point of 6.87.The protein contains a conserved RGS domain(IPR016137)and a Phox-associated domain(IPR003114),a Phox homologous domain(IPR001683)and a sorting nexin in C-terminal(IPR013937).Clustering analysis showed that MAPK proteinsfrom different filamentous fungiclustered into two clades,these MAPK proteins from three NTF were clustered in a single clade,suggesting that they share a close evoltutionary relationship with each other but distant from other fungi.Similarly,RGS4 proteins from three NTF were also clustered in a single clade,and they showed a distant evolutionary relationship with other RGS4 proetins.2.Knockout vectors of two genes were recombinant through Saccharomyces cerevisiae methods,and obtained positive yeast transformations.The full length fragment of target gene and hph cassettewas introduced into the protoplast of D.haptotyla,and the positive transformants were screened using hygromycin.The knockout strains of Mapk and Rgs4 genes were verified by PCR and Southern blot,respectively.3.Comparison on trap formation,spore production,the growth rate and the resistance to stress between the wild type(WT)and mutants.Our results showed that MAPK protein involved in trap formation,spore production,the hyphal growth and differentiation,and the resistance to stress,especially,MAPK protein plays a very important role in the trap formation and spore production.The Mapk knockout strain did not produce traps and conidia;RGS4 protein involved in trap formation and spore production,it had little effect on mycelial growth and differentiation and stress resistance,the number of conidia and predatory organs were significantly decreased.Innovations in this paper:The Mapk and Rgs4 genes of D.haptotyla were knocked out and their phenotypes features of the two mutants were compared with those in the WT strain.According to those results,functions of MAPK and RGS4 proteins in D.haptotyla were preliminarily analyzed.