假禾谷镰孢酵母双杂交文库构建及FpgBTB1互作蛋白筛选OA

[Objective]To construct a high-quality cDNA yeast two-hybrid(Y2H)library of Fusarium pseudograminearum to obtain proteins that directly interact with FpgBTB1 in F.pseudograminearum.[Method]The mycelia of F.pseudograminearum strain WZ-8A were used as the material,for total RNA extraction.Gateway technology was used in the construction of the primary library and the pri-mary library plasmid was connected with the pGADT7-DEST vector through LR recombination reaction to obtain the secondary library of the nuclear system.The bait recombination vector of FpgBTB1 was constructed by homologous recombination,and Y2H co-transformation was used to screen its interact-ing proteins,followed by one-to-one interaction verification.[Result]The capacity of the primary library and secondary library were 1.20×107 CFU and 1.40×107 CFU respectively.The size of the inserts ranged from 750 bp to 2 000 bp,and the recombination rate reached 100%.The recombinant bait vector pGBKT7-FpgBTB1 was successfully constructed,and six candidate interacting proteins were obtained through the Y2H library.One-to-one verification results showed that fructose-1,6-bisphosphate aldolase(XP_009261710.1),tubulin alpha chain(XP_009257790.1),superoxide dis-mutase(XP_009261302.1),and a protein containing endonuclease/exonuclease/phosphatase domains(XP_009256684.1)interacted with FpgBTB1 protein.[Conclusion]A high-quality cDNA library of F.pseudograminearum was constructed,and four proteins interacting with FpgBTB1 were successfully identified,which laid a foundation for revealing the function of FpgBTB1 in F.pseudograminearum.