| Pinus massoniana is a pioneer tree species for afforestation in barren areas of south China because of its advantages of fast growth,drought tolerance and poor adaptability.Pine wilt disease is a devastating disease of pine caused by Bursaphelenchus xylophilus,which seriously threatens the healthy growth of P.massoniana.At present,some genes related to disease resistance have been found in the study of resistance mechanism of P.massoniana to B.xylophilus invasion.In the previous study,the laboratory obtained several resistance genes of P.massoniana in response to the infection of B.xylophilus,among which DRG(disease resistant gene)gene was inhibited in P.massoniana with B.xylophilus infestation,which was speculated to be related to the disease resistance of P.massoniana.In order to deeply investigation on the correlation between the gene expression and host resistance to B.xylophilus infestation,this study cloned the CDS of DRG in P.massoniana,to construct a recombinant expression vector of DRG fusion with GFP gene and thereafter genetically transformation into model plant Arabidopsis thaliana.PWN disease resistance of transgenic A.thaliana and the control line were comparatively study,to explore the regulation of DRG on disease resistance.The main results are as follows:DNA sequencing and homology alignment of DRG gene from P.massoniana showed that the CDS of DRG gene contains 624 bp,which encodes 207 amino acids;and the DRG gene is almost consistent with that of t Avr9/ CF-9 rapidly elicited gene from Pinus sylvestris(Ps ACRE),with three nucleotides and an encoded amino acid different.,These results indicated that the two genes had high homology,which suggested that this ACRE gene is relatively conserved among different pine varieties.Therefore,this gene was regarded as ACRE gene in P.massoniana(PmACRE).The recombinant expression vector of PmACRE gene fusion GFP was constructed to genetically transform into A.thaliana.Subsequently,the positive transgenic A.thaliana was identified by PCR amplification of PmACRE from genomic DNA,green fluorescence observation with fluorescence microscopy,and Western-blotting determination of ACRE fusion protein expression.The results of microscopic examination and phenotypic observation showed that the nematodes could migrate from the wound site to the leaves and cause the leaves to turn yellow and wither,indicating that the nematodes successfully infected the A.thaliana.By comparing the incidence of transgenic A.thaliana with PmACRE gene and the control group inoculated with different nematode populations,the results showed that the pathogenicity of B.xylophilus to transgenic A.thaliana with PmACRE gene was lower than that to the control group.The pathogenicity rate of transgenic A.thaliana with PmACRE gene was 28%-46%,while that of control group was 37%-59%.The results indicated that heterogeneous expression of PmACRE in A.thaliana improved the defense ability of plant against B.xylophilus infection.In addition,after inoculation for 1,2,3 and 5 days,the contents of phytoalexin such as total phenolics and total flavonoids in leaves of transgenic A.thaliana,and capacity of ascorbate peroxidase were higher than those in control group.Furthermore,the interaction proteins of ACRE were captured by GFP-Trap CoImmunoprecipitation(CO-IP)technique,and the regulatory network of ACRE proteins in A.thaliana was studied.The results of mass spectrometry identification and protein function analysis showed that,ACRE protein and ATP synthase subunit alpha,beta subunits and serine hydroxymethyl transferase,catalase domaincontaining protein proteins,myrosinase,Dihydrolipoyl dehydrogenase,Ketol-acid reductoisomerase,Geranylgeranyl diphosphate reductase,chloroplastic,Sadenosylmethionine synthase,glutamine synthetase,and many other proteins interact with ACRE.GO annotation of the interacted proteins showed that these proteins in the cellular component is a part of the most cytoplasm,plasmids,envelope,nonmembrane-bounded organelles and apoplast,participate in most of the molecular function is combined with the function and the catalytic activity,biological processes involved in the most cell process,response to the reaction of inorganic and metal ions.KEGG enrichment analysis showed that the significantly enriched pathways were metabolic pathways,followed by biosynthesis of secondary metabolites and carbon metabolism pathways.Among them,serine hydroxymethyl transferase,S-adenosine methionine synthase,hydroxylase,and glutamine synthase play an important role in the regulation of stress tolerance and metabolism.In conclusion,the results of this study suggest that PmACRE gene can regulate resistance to B.xylophilus infection in A.thaliana,and enhanced the activity of PAL in A.thaliana,promoted the synthesis of flavonoids and phenolic compounds,and improve the resistance and defense ability of A.thaliana.ACRE proteins regulate the synthesis of phytoalexin,which facilitates to remove the toxic substances and improve the disease resistance of A.thaliana by interacting with multiple proteins in the secondary metabolic pathway,detoxification resistance pathway and primary metabolic pathway.These results provide a theoretical basis for discovering functional genes that effectively regulate resistance to B.xylophilus disease in P.massoniana,and provide a reference for revealing the complex interaction mechanism between B.xylophilus and its host plant. |
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