| Ubiquitin proteasome pathway(UPS)is a protein degradation pathway in eukaryotes,which is realized by four parts: ubiquitin activating enzyme E1,ubiquitin binding enzyme E2,ubiquitin linking enzyme E3 and 26 S proteasome complex.There are four types of plant ubiquitin ligase E3: anaphase promoting complex(APC),HCT to E6-AP C terminus,SCF(a complex of Skp1,Cdc53,and F-box protein)and ring(really interesting new gene)/ U-box.Among them,plant U-box protein is simply called PUB(plant U-box protein).It has been reported that there are at least 64 PUB gene members in Arabidopsis.Some studies have shown that Arabidopsis PUBs(AtPUBs)family members play an important role in plant stress response,and many family members are complementary or redundant.AtPUB10 is a member of the AtPUBs family.It inhibits the jasmonic acid(JA)pathway and negatively regulates abscisic acid(ABA)by mediating the ubiquitination and degradation of MYC2 transcription factor of the basic helix loop helix family.However,there is a lack of response of pub10 mutant to plant pathogens,and whether AtPUB11,which is highly homologous with AtPUB10,is involved in the regulation of plant pathogen response.In order to analyze the relationship and homology between Arabidopsis E3 ubiquitin ligases PUB10 and PUB11,bioinformatics analysis was performed on AtPUB10 and AtPUB11.We identify the genetic material of the Arabidopsis pub10/pub11 double mutant,from the aspects of PstDC3000 infection,pathogenic bacteria-induced changes in plant mutant MAPK,flg22 stimulation of Arabidopsis leaf ROS release,and quantitative immune gene expression levels,etc.,explore AtPUB10,AtPUB11 in resisting PstDC3000 regulation.According to this research idea,this paper carried out relevant experiments,and the specific research results were as follows:(1)Through bioinformatics analysis,it was found that the constructed AtPUBs family evolutionary trees AtPUB10 and AtPUB11 were clustered together.The similarity of the amino acid sequence alignment of the two proteins reached 75.44%.The gene promoter has various light response elements,hormone response elements and defense Like stress response elements,the secondary structure is mainly composed of ?-helix and random coil,and the tertiary structure is very similar,indicating that the two have a very close relationship and homology,which may be similar in Arabidopsis Biological function.(2)In order to further analyze the biological functions of AtPUB10 and AtPUB11,the triple primer method was used to identify pub10/pub11 double mutant and verified by detecting their expression in the mutants;constructing p DT1-PUB10 and p DT1-PUB11 vectors,and through Agrobacterium transformation obtained transgenic materials;constructed the p GEX4t-1 vector of the PUB10 gene segment,and expressed and purified the PUB10 segmented protein and obtained the PUB10 antibody by immunizing mice with the GST-PUB10-ARM segmented protein,It lays the foundation for carrying out protein experiments to verify the biological functions of AtPUB10 and AtPUB11.(3)Through experiments of plant resistance to pathogenic bacteria,it was found that the pub10/pub11 double mutant was more sensitive to PstDC3000.Further analysis of the effects of AtPUB10 and AtPUB11 deletion on plant immunity revealed that the MAPK phosphorylation level of the pub10/pub11 double mutant after PstDC3000 treatment was Rapidly decreased between 10-20 minutes,ROS release decreased after flg22 stimulation,and JA expression genes and negative immunoregulatory genes increased significantly,indicating that AtPUB10 and AtPUB11 have positive regulatory effects against PstDC3000.To sum up,the results of this paper show that AtPUB10 and AtPUB11 have a positive regulatory role in the process of resistance to PstDC3000 pathogens,and there is functional redundancy. |
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