Screening Of NbMORC3 Interacting Proteins And Cultivation Of Transgenic Plants

NbMORC3 was isolated by methylation sensitive amplification polymorphism(MSAP)in previous research to clone genes related to cadmium stress in our lab.NbMORC3 belongs to MORC(microrchidia)family which is a highly conserved nuclear protein superfamily that regulates downstream gene methylation and transcription,and participates in epigenetic processes such as chromatin condensation and remodeling.The well-studied MORC members have conserved N-terminal GHKL-ATPase,C-terminal CC(coiled-coil)domain and zf-CW domain,while NbMORC3 has no C-terminal CC domain.It is still unknown whether NbMORC3 shows the similar functions of reported MORC members,and more is elusive of its roles in regulation of plant development.The ATPase activity of truncated NbMORC3 was analyzed by prokaryotic expression technique.The intermolecular interaction of NbMORC3 was analyzed by bimolecular fluorescence complementation(BiFC).The subcellular localization of NbMORC3 was observed by laser confocal microscope.And the interacting proteins of NbMORC3 was screened from Nicotiana benthamiana c DNA library by yeast two-hybrid Lex A system.The NbMORC3CRISPR and overexpression transgenic plants were cultivated by Agrobacterium-mediated transformation.The main results are as follows:(1)Prokaryotic expression:ATPase domain of NbMORC3 protein was inducd to express in E.coli.The truncated protein was solved in supernatant of extraction buffer.Analysis of ATPase activity showed that the truncated NbMORC3 had the similar catalysis of reported MORC proteins.(2)Bimolecular fluorescence complementation:the full length of NbMORC3 was fused to the C-terminal and N-terminal of YFP semi-molecule respectively.The YFP fluorescence was observed by confocal microscopy after 60 hours of agrobacterium co-infiltration of BiFC constructs in Nicotiana benthamiana leaves.The results showed that there were no YFP fluorescence observed for NbMORC3 constructs coinfiltration instead of positive control,which suggested that there were no intermolecular interaction between NbMORC3 proteins.(3)Subcellular localization:the full length of NbMORC3 was fused with YFP protein,and transiently expressed in N.benthamiana leaves.The results showed that NbMORC3 was localized in the nucleus.(4)Screening of interacting proteins by yeast two-hybrid:the bait vector of NbMORC3 was constructed,then its cytotoxicity and self-activation activity were detected in yeast strain SKY48.The results showed that there were no self-activation of NbMORC3in lex A system and no harm to cell growth in SKY48.Eventually total of 15NbMORC3-interacting proteins were screened by Y2H.It has been proved experimentally that NbBIN4 and NbMORC3 can interact in the nucleus.(5)Genetic transformation of Nicotiana benthamiana:the cultivation of NbMORC3CRISPR and overexpressed transgenic plants were mediated by Agrobacterium tumefaciens.There were 20 of T₀generation overexpression positive seedlings and 15 T₀generation CRISPR positive vaccines obtained after molecular verifications were carried out respectively.In this study,the function of ATPase domain and the sublocalization of NbMORC3were explored.The NbMORC3-interacting proteins were screened,and the transgenic plants were cultivated.Our study will lay a foundation for further research on the roles of NbMORC3 in regulation of plant development.