Functional Analysis Of Pathogenic Candidate Gene Pgmiox In Pyrenophora Graminea

In this study,RNA interference technology was used to perform a functional study on the gene Pgmiox of Pyrenophora graminea.The gene was cloned from the wild QWC strain and its RNA interference vector was constructed.After PEG-mediated genetic transformation,54 transformants were screened by hygromycin resistance.6 interference mutant strains were randomly selected for functional analysis,and the functional mechanism of pathogenicity of Pgmiox gene in Pyrenophora graminea was identified.Lay the foundation of the gene regulation mechanism and pathogenic mechanism of bacteria.The main conclusions of this study are as follows:1.The Pgmiox gene of Pyrenophora graminea was cloned and analyzed by bioinformatics.The results showed that the gene had no intron,the Pgmiox is a gene of length 981 bp and encodes 326 amino acids.The gene was submitted for registration in GenBank with the sequence number of MK302448.The software analyzed the isoelectric point of the protein encoded by the gene to be about 4.93,and the protein was an acidic protein;The secondary structure of the protein was predicted,with ? helix(Alpha helix,Hh)accounting for 46.93%;Protein domain analysis showed that the gene has a miox domain in the Pfam domain;The phylogenetic tree constructed was concluded that the protein sequences of the inositol oxygenase gene of P.graminea and Pyrenophora tritici-repentis were combined together.2.Interfering fragment Pgmiox1,fragment Pgmiox2 and vector pSilent-1 were used to successfully construct the RNA interference vector pSilent-1Pgmiox of the Pgmiox gene.The protoplasts of wild strain QWC were prepared.After PEG4000 mediated genetic transformation,54 transformants were obtained by hygromycin resistance screening.6 interfering mutants ?Pgmiox32,?Pgmiox33,?Pgmiox35,?Pgmiox43,?Pgmiox44 and ?Pgmiox54 were randomly selected,and the interference rates were determined by real-time fluorescent quantitative PCR,which were 90.36%,89.87%,88.47%,90.56%,90.75% and 88.03% respectively.3.The functional verification analysis showed that the interference mutation of Pgmiox gene significantly affected the vegetative growth of P.graminea;Pgmiox was related to the response of P.graminea to different environmental stresses;the gene was involved in the regulation of cell wall integrity of P.graminea;in the test of resistance to 4 fungicides.The Pgmiox gene enhanced the sensitivity of P.graminea to iprodione and prochloraz.Under the stress of tebuconazole and carbendazim,the growth rate of interference mutants increased significantly,and the growth rate was faster than that of the control QWC strain.The tolerance of mutants to these two agents increased.This gene may be the target site of tebuconazole and carbendazim.In the toxin test,the toxins induced by the 6 interfering strains were significantly different in color from the wild strain QWC.The toxin liquid of the wild strain was dark brown,and the interfering strains liquid were generally pale yellow.The toxin liquid was injected into the leaves of the same part of potted barley.The leaves injected with the toxin from the QWC strain were completely diseased,and the leaves shriveled and became dark brown.The leaves injected with the toxin of the interfering strains were significantly weakened,and the leaves were green overall,and only the injection site was brown.The Pgmiox gene is involved in regulating the pathogenicity of P.graminea toxin.In the pathogenic pot test,the incidence of 6 interference strains were significantly lower than that of the control QWC strains.Barley infected by the QWC strain "sandwich method" showed weak growth,severe leaf disease and leaf atrophy.The dried barley was yellow-brown.Barley infected by the interfering mutants grew stronger,the disease was significantly weaker,the leaves were yellow-green,and there were fewer disease spots.In the pathogenic in vitro test,the leaves of the wild strain QWC fungus cake infected the leaves,and the leaves were significantly diseased,showing a yellow brown lesion.The leaves infected by 6 interfering strains were significantly weaker,and the fungus cake was infected.It was pale yellow and no lesions were formed.The Pgmiox gene is involved in regulating the pathogenicity of P.graminea.