Utilizing Y2H-seq to screen interacting proteins of rice OsTLP16.5

To screen for interacting proteins of the rice thylakoid lumen protein OsTLP16.5 (encoded by Os06g0705100), which will help to elucidate the molecular regulatory mechanisms of OsTLP16.5 in rice. Total RNA was extracted from rice to construct a cDNA library. A yeast two-hybrid (Y2H) bait vector, pGBKT7-OsTLP16.5, was constructed to screen candidate target proteins using the yeast two-hybrid system, followed by interaction validation. Large-scale screening was performed using next-generation sequencing (NGS) technology. The bait vector pGBKT7-OsTLP16.5 was successfully constructed and verified to be free of autoactivation. A library screen identified 32 potential interacting proteins, of which 3 showed positive interactions after validation. NGS technology revealed 977 potential interacting proteins, including 51 chloroplast-associated proteins. A rice cDNA library was successfully constructed, and interacting proteins of OsTLP16.5 were identified, providing a foundation for studying how OsTLP16.5 functions in rice.