The Function Analysis Of Metalloprotease Gene In Metarhizium Robberstii

Entomopathogenic fungi is one of the biggest groups in insect pathogenic microorganism groups,and its number had got to nearly 1000 kinds reported by all around the world,as well as,it is the one of the main factors in controlling insect populations in nature.As an important entomopathogenic fungi,Metarhzium spp.has been widely exploited for biological control of insect pests.However,due to the traditional Metarhzium spp.pesticides have the following disadvantages,including the slow speed of kill of insects and longer pathogenic porcess,the big dose used in field,the high cost,and environmentally sensitive,which restricts the further large scale of its application.Infection insects by Metarhzium spp.is a relatively complex processes,in which proteases play a major role in this processes and are considered as a main virulence factor.The current research is mainly involved in subtilisin-like proteases(Pr1)and trypsin-like proteases(Pr2)in Metarhzium spp.Metalloproteinases is one kind of secrete extracellular protease in early infection of insects,which is also considered as a kind of key virulence factor.It have been reported that the expression of gene metalloproteinase 1(Mamp1,EFY97549)significantly increases in M.robberstii after heat shock.Therefore,we speculated that gene Mamp1 play an important role in the process of heat-resistant spore under heat stress [1],moreover,gene function of the Mamp1 as a virulence factor has not been verified by gene knockout.To insight into Mamp1 function,WT(wide type strain)M.robberstii as experimental material,using of Rapid amplification of cDNA ends(RACE)technique to get the 5,and 3,untranslated region and its whole CDS sequence for the bioinformatics analysis of the gene.Afterthat constructed the knockout vector pDHtBar-K constains the sequence of gene Mamp1 by choosing the plasmid pDHt-SK-Bar through digestion,ligation and PCR validation.We conducted Agrobacterium-mediated transformation(ATMT)and homologous recombination technology and co-cultivation with M.robberstii to realize the knockout of the gene Mamp1.At last,growth rate,sporulation,stress resistance and insect bioassay and other phenotypic differences were used to determine the exact function of the gene by the comparion of wild strains and the mutant strains.The main results are now stated as follows:1 ? The CDS structure and analysis of metalloproteinases gene in Metarhzium.Sequence analysis show that gene Mamp1 contains a 1942 bp open reading frame(ORF),flankedy by a complete 93 bp 5,untranslated region and 105 bp 3,untranslated region.The ORF encodes a putative 611 amino acid and its calculated molecular mass is 68 KDa and pI 6.17.BLAST analysis show that there is a highly homology for the gene and other entomopathogenic fungi.2?The function of gene Mamp1 in Metarhzium.(1)Bioassay experiments of infection wax moth show that median lethal time(LT50)values for knockout strains were calculated as 8.77?7.8 and 5.4d when the concentration of spores is 5×106,1×107 and 2×107 spores mL-1,respectively.Compared to wild-type parent,LT50 values increased by 19%,23.7% and 24%,respectively.These results indicated that the ability of infection wax moth has decreased to the ?MP1 strain and its virulence has decreaded significantly.Therefore,gene Mamp1 plays an important role in the virulence of Metarhzium spp.(2)Stress experiments show that the mutant strain to high temperature(38?)and UV resistance significantly decreased,spore germination rate decreased by about 13.9% and 18.5%,respectively;Responses to several other chemical substances stress including cell wall–disturbing agents substances SDS and Congo red,oxide-type regulating substances,H2O2 and menadione(MND),osmotic adjustment substance NaCl,sterilization substances carbendazim(CBD),the diameter of colony growth of knockout strains had different decreases,the data were approximately 13.4%,8.4%,3.6%,6.2%,16.9%,10.8% compared with the wild-type strain.Therefore,metalloprotease gene plays a role in the process of stress resistance in Metarhizium.(3)The experiment of growth and development show that the growth rate on PDA and PPDA medium was no significant difference between wild-type strain and konckout strain.However,the growth rate of knockout strains represented an increase of about 12% and 12.34% with the wild strain on SDAY and 1/4 SDAY medium.In the measurement process of sporulation,there is no significant difference in sporulation between PDA and 1/4 SDAY medium showed in the final results,while on the SDAY medium,the knockout strain had about 15% reduction in conidia production than the wild strain.Therefore,metalloprotease gene had less effect on the growth and development of fungi.Gene function of metalloproteinase-1 were preliminary illustrated in the present study,the results not only help us understand in depth the mechanism and the mechanism of stress tolerance of metalloproteinase-1 gene in infection of insects by Metarhizium,but also provide a theoretical basis for the further utilization of metalloproteinase to create engineering strains.