Screening And Identificating Of Proteins Interacting With OsMKK1 And OsMKK6 In Rice

The plant MAPK cascade pathway is widely involved in the regulation of cell differentiation,hormone response,stress response,growth and development in plant.OsMKKs are the mitogen-activated protein kinase kinase proteins in the midstream of MAPK cascade,which converges the upstream signals and delivers the signals to downstream proteins in the pathway,to increase the plant adaptations to external adverse environments.Previously,OsMKKs family proteins were found to be divided into four subgroups A,B,C and D,OsMKK1 and OsMKK6 were the two subgroup A members.In addition,OsMKK1 was signaling in the salt stress,OsMKK6 was in cold stress signaling at the seedlings of rice.To further study the regulational function of OsMKK1 and OsMKK6 in MAPK signal transduction pathway,in this study,A large-scale screening of many proteins interacting with OsMKK1 and OsMKK6 was performed in yeast two-hybrid rice system of rice cold stress cDNA library.Meanwhile,mutant lines of OsMKK1 and OsMKK6 by CRISPR/Cas9 technology were developed and identified by qRT-PCR.The main results are as follows:In order to screen proteins interacting with OsMKK1 and OsMKK6,two bait vectors were constructed?pGBKT7-MKK1 and pGBKT7-MKK6?and the self-activating activity was tested.The results show that,pGBKT7-MKK1 and pGBKT7-MKK6 were detected to have non-toxic effects to yeast growth and have no self-activating activity.After repeated screening of selection media SD/-Leu-His-Trp,SD/-Leu-His-Trp-Ade and SD/-Leu-His-Trp-Ade+X-?-Gal,and 40 positive clones were selected to interact with MKK1 and 118 positive clones selected for MKK6 interactions,the point-to-point verification was conducted by co-transformation.Only four proteins?OsATP-S,OsGDSL,OsSyt-2 and OsPPase?were identified to interact with OsMKK1,and five?OsSyt-3,OsAE7,OsCIPK17,OsGAPDH and OsKIN10?proteins interacted with OsMKK6.Bimolecular fluorescence complementation?BiFC?assay showed that:OsATP-S and OsGDSL were interacted with OsMKK1 in rice protoplasts;OsSyt-3,OsAE7,OsCIPK17 and OsGAPDH were interacted with OsMKK6 in rice protoplasts.The CRISPR/Cas9 gene editing technique was used to construct the dual-target vectors of the OsMKK1 and OsMKK6 genes.The grobacterium-mediated transformation system was employed to obtain transgenic rice plants for positive plants screening by Cas9 gene PCR detection,6 lines for the CRISPR-MKK1 target and 8 lines for CRISPR-MKK6 target were obtained.PCR detection and sequencing of the editing site showed that 5 CRISPR-MKK1 lines and 7 CRISPR-MKK6 lines obtained positive seedling target sites.2 in 5 lines for the CRISPR-MKK1 target were the homozygous plants with cutting off 500 bp gene fragment,the rest plants are point mutation plants.In addition,the CRISRP-MKK1 target transgenic plants have all harvested T₁ generation seeds.qRT-PCR assays showed that:the gene expression levels of the transgenic homozygous lines of OsMKK1 was only about 20%of the wild-type,the transgenic heterozygous lines of OsMKK6 expressed only about 40%of the wild type.These result indicated that the gene-edition was successful.These transgenic lines could be used in the regulatory mechanisms study of OsMKK1 and OsMKK6 in rice abiotic stresses.