Study On Functions Of A Collagen-like Protein Gene (Mcl2) From Metarhizium Robertsii

Entomopathogenic fungi have wide and great potential for developing as microbial control products to control insect pests that cause significant economic losses in agriculture and forest,because of their environmental security,high insect specificity and potential epidemic.Metarhizium robertsii,as a model organism of entomogenous fungi,has been at the forefront of studying genetic regulatory mechanism,pathogenesis and biological control.The collagen-like protein,which exists in the viruses,bacteria and eukaryotes,acts as a crucial part in the regulation of the growth and development,metabolic pathways,pathogenicity and other aspects of many organisms.Previous studies of our laboratory showed that the increased rate of a collagen-like protein gene was the top three in all up-regulated expression genes according to the transcriptome expression profile analysis of wild-type M.robertsii conidia treated with heat shock.Therefore,it has been speculated that the collagen-like protein plays a very important role in the process of stress resistance in Metarhizium.Here,systematic studies on functions of the collagen-like protein(Mcl2)were performed in M.robertsii.Our results will be helpful to clarify the genetic information of Mcl2 and identify the mechanism of growth and development,stress resistance and the ability of insecticidal effect,also to provide a new reserve for the artificial creation of effective insecticidal transgenic Metarhizium strains.Firstly,Mcl2 was cloned and annotated,and the genetic information of the gene was obtained.Then,the knockout and complementary strains were generated by employing the method of genetic transformation mediated by Agrobacterium tumefaciens.Finally,the functions of Mcl2 were investigated through comparing the phenotypic characteristics between the wild type,Mcl2 deletion and complementary mutants.The results are as follows:(1)The full length cDNA sequence of Mcl2 gene and its biological information were acquired by using the RACE,sequencing and annotating.And the results indicate that Mcl2 from Metarhizium robertsii may be a new collagen-like protein gene through Blast alignments in GenBank database and phylogenetic tree.(2)By building pDHt-SK-Mcl2L-BAR-Mcl2 R and pDHt-SK-Mcl2-BEN recombinant plasmids,A.tumefaciens-mediated and homologous recombination gene knockout method,the ?Mcl2(knockout mutant)and ?Mcl2/Mcl2(anaplerosis mutant)strains were successfully gained.(3)Comparisons of the biological traits between WT(wild type strain),?Mcl2(knockout mutant)and ?Mcl2/Mcl2(complementary mutant)indicated that there were no significant differences in the color and morphology of mutants colony,mycelial growth rate between the knock-out and wild-type strains on the PDA,PPDA,SDAY and 1/4 SDAY mediums.Howerer,the sporulations on SDAY,1/4SDAY,PPDA and PDA mediums are slightly lower than the wild type strains,decreased by8.34%,9.27%,8.45% and 9.89%,respectively.The sporulations of ?Mcl2/Mcl2 are basically in line with WT.(4)Conidia treated with heat shock 38 °C for 2 hours of the three strains,stress analysis showed that the conidia germination rate of deletion strains reduced to40.19%.Compared with the wild-type strains,it had reduced 34.79%,which showed the deletion strains were more sensitive to changes of the outside temperature.However,treated with the ultraviolet irradiation for 3 min,conidia germination rate of knockout strains was 56.48%,compared with wild-type strains and complementary mutants,9.45% and 9.10% were reduced,which did not change remarkably.Moreover,there were no indistinguishable difference in the growth on the PDA added NaCl,H2O2,SDS,Congo Red and other chemical substances in chemical stress study,respectively.(5)Infecting wax moth with three strains through the condidia concentration 107conidia/mL.Compared with the WT,the infections of the deletion mutants were significantly reduced.The fifth day's mortality rate dropped 49.74%.Furthermore,the results indicated that the half death time for wax moth was 7.47±0.7 days,and prolonged two days.It suggested that the deletion of Mcl2 resulted in the decreasing of virulence.In summary,the lack of the gene of collagen-like protein had not a great influence on the growth and development,chemical stress,UV and sporulation in M.robberstii.However,our data show that the gene Mcl2 may play an important role in the ability to adapt to the heat stess environment and insecticidal capacity.