Functional Study For The Gene (Xrn1) Of 5'-3' RNA Exonuclease In Metarhizium Robertsii

Metarhizium robertsii is a filamentous fungus that can be used for both plant diseases and insect pests.Like other entomogenous fungicides,biofungal insecticides based on Metarhizium spores have defects such as unstable insecticidal effect and short shelf life;Therefore,it is necessary to conduct in-depth research on the growth and development,pathogenicity and stress resistance mechanism of pathogenic fungi such as Metarhizium to provide a theoretical basis for the commercial development of Metarhizium and the improvement of the biocontrol effect of the bacteria.RNA processing and degradation are important steps in regulating gene expression,and play a vital role in regulating the growth and development of eukaryotes.The degradation of non-coding RNA depends on the action of 5'-3' exonuclease,of which 5'-3' RNA exonuclease?Xrnl?is an important factor related to RNA metabolism and RNA interference A large family of functionally related conserved enzymes exists in all eukaryotes.Previous reports have found that Xrn1 plays an important role in the growth and development and pathogenicity of filamentous fungi such as Fusarium graminearum.However,there are few reports on insect pathogenic fungi including Metarhizium.Therefore,this study used Metarhizium robertsii as the research object,and conducted an in-depth study on the biological function of MrXrn1?MAA₀9154?in the process of growth and pathogenicity of Metarhizium,First,bioinformatics analysis was performed on the sequence characteristics of MrXrn1;secondly,the knockout strains?MT?and supplementary strains?CP?obtained by using gene knockout technology,Agrobacterium-mediated genetic transformation technology combined with biochemical experiments,etc.The biological characteristics?including growth and development,stress resistance and virulence?of wild type strains?WT?were compared and analyzed to clarify the biological function and possible regulatory mechanism of MrXrn1.The specific research results are as follows:1.MrXrn1 sequence feature analysis:Searching in NCBI to obtain the full-length sequence of MrXrn1,encoding 1419 amino acids;the results of conservative functional domain analysis found that there is a conserved XRN_N domain at its N-terminus?Accession:pfam 03159?.Further construction of the phylogenetic tree revealed that it has a high affinity with Metarhizium acridum and Metarhizium rileyi Xrn1.2.Acquisition of MrXrnl knockout and supplementary strains:According to the genomic location of Xrn1?MAA₀9154?,select the flanking sequences and full-length sequences at both ends;construct its knockout vector plasmid and complement vector plasmid,and obtain MT and CP by Agrobacterium-mediated transformation method and homologous recombination method.3.Analysis of the role of MrXrn1 in the growth and development of Metarhizium:Cultivate different strains?WT,MT,and CP?under the same conditions,observe the color,size,and morphology of the colonies,simultaneously determine the colony growth diameter,and calculate and analyze the spore production of different strains;at the same time,regularly observe the germination rates of WT,MT,and CP,Calculate when the germination is in progress.It was found that compared with WT/CP,MT spore production decreased by 80.91%,and spore germination was delayed by 2 hours.In addition,compared with WT/CP,MT has no obvious difference in colony color,shape and size;4.Analysis of the role of MrXrn1 in the stress of Metarhizium:Different strains?WT,MT and CP?were subjected to stress analysis;the spore suspensions of different strains were treated with water bath and ultraviolet irradiation at 40?,45?,respectively,and placed on PDA medium.The results show that:Compared with WT/CP,the relative germination rate of MT decreased by 73.10%under the treatment condition of 45?,these results indicate that the knockout strains are more sensitive to heat shock?decreased heat tolerance?;The relative germination rate of MT was reduced by 24.96%under these conditions.These results indicate that the knockout strains are more sensitive to ultraviolet stress;In addition,under the treatment conditions of chemical reagents?SDS,NaCl,Congo Red,H2O2,Menadione?,the relative inhibition rate was used as an index?calculated by the growth diameter of different strains?.There was no significant change in the relative inhibition rate.5.Analysis of the pathogenic role of MrXrn1 in Metarhizium:The bioassay was conducted on different strains?with Galleria mellonella as the test insect?.The results showed that in the body wall infection test,compared with WT/CP,the MT-infected wax borer had a higher survival rate.Further calculating the half-lethal time?LT50?,these results indicate that MrXrn1 knockout results in a significant decrease in virulence.In the direct injection inoculation test,there was no significant difference in the survival rate of the wax borer after infection with different strains.Based on these data,we speculate that MrXrn1 may play an important role in spore adhesion or body wall penetration of fungi.Further analysis of the mechanism of MrXrn1 knockout leading to a decrease in virulence revealed that the ratio of spore formation of MT on the hydrophobic surface and cicada wings was reduced by 38.24%and 40.64%,respectively,compared with the control group WT/CP.In summary,the study found that MrXrn1 spore production decreased significantly,the germination time was relatively delayed,the sensitivity to temperature changes and UV increased,the virulence weakened,and the attached spores decreased.These results indicate that MrXrn1 plays an important role in regulating Metarhizium growth,stress resistance and pathogenic processes.The results of this study provide a basis for exploring the field application of Metarhizium.