Preliminary Study On The Function Of GPCR (G Protein Receptor) Related Genes In

Nematode-trapping fungi is one of the most important factors to adjust the density of nematodes in natural environments. Arthrobotrys oligospora is one of the best-studied nematode-trapping fungi, the interaction between nematode-trapping fungi and nematodes is also researched by A. oligospora that is known as a model fungus. Trapping devices （traps） are the important tools for fungi to capture nematodes, and the formation of traps by nematode-trapping fungi is an important mark of their change from the saprophytic to the predacious lifestyles, however, little is known about the molecular basis of the trap formation in the nematode-trapping, fungi. In this study, eleven GPCR proteins （AOL_s00054g15、AOL_s00169g44、 AOL_s00054g207、AOL_s00075g223、AOL_s00076g24、AOL_s00210g301、 AOL_s00215g301、AOL_s00006g420、AOL_s00097g558、AOL_s00043g705、 AOL_s00043g768） were functionally analysed by gene knockout technology. GPCRs that are located in the upstream of signaling system, are a variety of multifunctional proteins that belong to superfamily. And they are the largest family of receptors on which located the cell membrane. Those proteins have a conserved seven a-helices transmembrane segment receptors domain, However, in the nematode-trapping fungi, the functions of GPCR have not yet been covered so far.The main obtained results were described as follows:1. Functional domains of the eleven GPCR proteins and cluster analysis. Most of the GPCR genes encode fewer amino acids （23-76 kDa）. They share one or more conserved domains except for 210g301. Among them,6g420 contains 7tm₂ super family, Git3_C super family and DUF3381 super family; 43g768 contains ABA_GPCR super family and DUF3735 super family; 97g558 contains 7tm₂ super family and Git3_C super family; 76g249 contains 7tm₂ super family and Git3_C super family. But, no conserved domain in 210g301 was predicted in GenBank and Interproscan web site. These four proteins contain more conserved domains than others, so their functions may be associated with multiple biological processes. The cluster analysis of fungal GPCR proteins indicates that many GPCR proteins are found in three species of nematode-trapping fungi, and the GPCR proteins in A. oligospora are clustered in four clades.2. Knockout vectors of the eleven GPCRs were built by electro-transformation of Saccharomyces cerevisiae and E. coli competent cells. A. oligospora protoplast was transformed by the method of CaCl2-PEG mediating transformation, and the method of PCR is used to screen and verify nine positive transformants. The nine mutants were compared with the wild type （WT） in growth rate, stress resistance, extracellaluar proteases production, sporophore morphology etc. The data indicated that the nine GPCRs are associated with those biological processes. There was change markedly in conidia production but mutant A15-4, the number of conidia were increased in A44-2 and A207-24, while other mutants （A215g-8, A223-2, A249-5, A420-25, A5’58-11 and A768-2） were decresed obviously in conidia production. Moreover, the traps were induced using nematodes and urea, the formation time and number of the traps were influenced by the knockout of the nine GPCR genes. The four mutants{A 15-4, A44-2, A558-4 and A 768-2) produced more traps, and A249-5 produced fewer traps, while other four mutans had no obviously difference from the wild type.3. According to our results, the nine GPCR knockout strains do not play a key role in growth, development, conidiation and trap formation in A. oligospora. The nine GPCR strains are the single gene knock out, these genes may have a complementary effect on the function, other GPCR genes may add to the part of the function of the deleted gene. Therefore, knock-out of the single GPCR gene, there are no significantly differences from the wild type in tested biological processes.Innovations in this paper:The nine GPCR-encoding genes of A. oligospora were triumphantly knocked out. Next the phenotypes and physiological features of the nine mutants were compared with those in the WT strain. Accoding to those results, functions of the nine GPCR were preliminarily presumed.