Cloning And Functional Analysis Of Potato E3 Ubiquitin Ligase StATL80

Potato(Solanum tuberosum L.)has become the fourth largest food crop in the world after wheat,rice and corn.Potatoes will suffer a variety of diseases during the growth process,seriously affecting the development of their industries.When resisting pathogenic bacteria,proteins will interact to complete the transmission of disease-related signals.Ubiquitin/26 S proteasome is an important protein degradation system that can remove junk proteins produced under stress conditions,thereby maintaining the normal function of cells.As an important part of the proteasome system,E3 ubiquitin ligase specifically recognizes its target protein and participates in the subsequent protein degradation process.In the laboratory,potato virus Y(Potato virus Y,PVY),Ralstonia solanacearum,and Phytophtora infestans were used to infect wild type potatoes,and multiple genes encoding E3 ubiquitin ligase were found to be affected high levels of induced expression.In this study,a gene with a high induced expression level was cloned and named StATL80;its function in resisting disease was studied.The main research results and conclusions are as follows:(1)Bioinformatics analysis of StATL80Design primers and amplify the StATL80 gene.The sequencing results show that StATL80 is 513 bp in length and encodes 171 amino acids.StATL80 has a close relationship with members of the ATL family of other species;therefore,it is speculated that StATL80 belongs to ATL family genes.(2)StATL80 has E3 ubiquitin ligase activity and participates in various stress responsesWhen StATL80 exceeds the standard,E3 ubiquitin ligase activity is the highest,indicating that StATL80 has E3 ubiquitin ligase activity.Tissue-specific analysis found that StATL80 was highly expressed in leaves.After hormone treatment,it was found that StATL80 was induced and its expression was significantly up-regulated.(3)StATL80 is located in the cytoplasm and nucleusObservation by confocal fluorescence microscopy revealed that GFP appeared in the nucleus and cytoplasm,indicating that StATL80 may be located in the nucleus and cytoplasmto play its biological function.(4)StATL80 gene negatively regulates plant disease resistanceTo verify whether StATL80 functions in the potato disease resistance process,it was found that the upregulated strain and wild-type potato were inoculated with PVX,PVY,Ralstonia solanacearum,and Phytophthora infestans.The symptoms of leaf yellowing and wilting caused by bacteria and the area of leaf brown lesions caused by Phytophthora infestans are more serious than those of wild type;while inoculated with PVY,PVX and Phytophthora infestans in down-regulated strains and wild-type potatoes,decreased the accumulation of viral CP in the strains,and the symptoms of leaf wilt and water stained brown lesions caused by Phytophthora infestans were lighter than those of wild-type potatoes;the above results showed that StATL80 gene negatively regulated potato virus resistance,bacterial wilt and potato infection of late blight bacteria.(5)There is an interaction between StATL80 and DSK2aFour proteins were obtained with StATL80 as bait.Verification revealed that the Ring ring domain of StATL80 and the central 1-4 domain of DSK2 a are the key domains for the interaction between the two.(6)Co-localization of StATL80 and DSK2 a in the nucleusSubcellular localization found that DSK2 a only exists in the nucleus;while StATL80 and DSK2a coexist in the nucleus,indicating that the two interact within the nucleus.In summary,StATL80 is a Ring-type E3 ubiquitin ligase,which is located in the nucleus and cytoplasm;StATL80 negatively regulates the resistance of potato to virus,Ralstonia solanacearum and Phytophthora infestans.The ring of StATL80 and the central 1-4 domain of DSK2 a are the key domains of the interaction between the two;but how does StATL80 affect the function of DSK2a? Can StATL80 ubiquitinate DSK2 a and promote its degradation by 26 s protease? These issues need to be further studied.