Construction of library of maize yeast two-hybrid induced by Spodoptera frugiperda feeding and screening of ZmRop1-interacting proteins

【Objective】To construct library of maize yeast cDNA induced by Spodoptera frugiperda feeding and bait vector encoding plant Rac protein，and to screen interacting protein from the library，with an aim to support further analysis on signal transduction mechanism of ZmRop1 in maize from the molecular level and improvement of maize resistance to S. frugiperda.【Method】Total RNA was extracted from different tissues of maize Zhengdan 958 fed by S. frugiperda， ds cDNA was synthesized by reverse transcription using SMART technology，and after linking to pGADT7 vector and transformed to yeast Y187，cDNA library of maize yeast two-hybrid was constructed. ZmRop1 was cloned with maize cDNA as a template to construct a bait vector pGBKT7-ZmRop1，which was transformed into yeast AH109 after enzyme digestion and sequencing identification，and the toxicity and self-activation activity of the bait protein were identified. Its potential interacting proteins were screened from the cDNA library by yeast two-hybrid with ZmRop1 as bait.【Result】The maize cDNA library obtained in this study had a capacity of 4.08×10⁶ CFU，library titer of 1.3×10⁸ CFU，good diversity of library fragments，and library recombination rate of 91.7%. The bait vector pGBKT7-ZmRop1 was successfully trans-formed into AH109 and identified as non-toxical and non-self-activating；22 proteins interacted with ZmRop1 were initially screened from the library，the analysis showed that they involved in regulating various biological activities such as plant defense.【Conclusion】The yeast two-hybrid cDNA library constructed in this study meets the requirements for library construction；the constructed ZmRop1 bait vector has no toxicity or self-activation activity；and 22 target proteins interacting with ZmRop1 are screened，which lays theoretical foundation for further study on ZmRop1-involved signal molecule mechanism of regulating plant defense and screening of insect-resistant genes in maize.