Study On The Mechanism Of Cytoplasmic Male Serility And Ferility Restoration In Brassica Napus Polima

Polima cytoplasmic male sterility(pol CMS)is one of the most widely used types of cytoplasmic male sterility in Brassica napus in my country,so it is of great significance to study the mechanism of its sterility and recovery phenomenon.Studies have found that the sterility gene of Polima cytoplasmic male sterility is orf224,and the restorer gene is Rfp,but how the sterility gene and restorer gene specifically cause the occurrence of cytoplasmic male sterility and the molecular mechanism of the restoration of fertility are still unclear.This study uses pol CMS sterile line,maintainer line,restorer line and high-generation near-isogene line materials constructed by sterile line and restorer line,through the method of reverse genetics and multi-omics joint analysis,to study the occurrence of pol CMS Preliminary analysis of the mechanism,the most important results of this research are as follows:1.Yeast two-hybrid screening of interacting proteins.The pol CMS restorer gene Rfp is a P-type PPR protein with mitochondrial guide peptide and 16 repeat motifs.According to its structural characteristics,4 bait vectors are designed to screen interacting proteins using yeast library.A total of 8 candidate proteins that interact with restorer genes were obtained;the sterile gene orf224 is a mitochondrial gene with two transmembrane domains.According to its structural characteristics,three bait vectors were designed to screen the sterile gene orf224 in the yeast library Through further yeast point-to-point experiments to confirm the four candidate proteins screened.2.Validate the selected candidate genes,and use the transient transformation expression system in tobacco leaves to design bi-molecular fluorescence experiment(BIFC)and luciferase(Luciferase)bi-molecular complementation experiments to verify that the restorer gene Rfp and the sterility gene orf224 were screened separately Of candidate genes.The study found that there is a multi-organelle RNA editing factor(MORF1)in the candidate gene of the restoration gene interaction,and the candidate gene of the sterility gene interaction also found a leucine-rich protein(LRR1)protein specific to anther development.The experiment found that these two proteins interacted strongly with the restorer gene and the sterility gene respectively in BIFC and Luciferase experiments.Then the prokaryotic expression of Rfp and Bna.MORF1 protein,using the in vitro protein interaction(pull-down)experiment found that the interaction of Rfp and Bna.MORF1 has been verified again.The Bna.MORF1-Flag label material was constructed by genetic transformation of Brassica napus,the total protein of Brassica napus was extracted,and the interaction between Rfp protein and Bna.MORF1 was confirmed again by co-immunoprecipitation(Co-IP).3.Subcellular localization candidate protein of protoplasts.The full-length CDS of Bna MORF1 and Bna LRR1 genes(without stop codons)was used to construct a subcellular localization vector of PM999-GFP,and the plasmid was transformed into protoplast by extracting protoplasts from Arabidopsis thaliana leaves Confocal microscope observation revealed that the interaction protein Bna.MORF1 of the Rfp protein and the interaction protein Bna LRR1 of orf224 were all located in the mitochondria.4.Use CRISPR/Cas9 technology to transgene verify candidate genes,and determine the expression levels of different copies by RT-PCR.It was found that Bna A01g0360 D copy expression was the highest in sterile line and reproductive line materials,and the other copies were weakly expressed,so CRISPR was used /Cas9 knocked out the candidate gene Bna.MORF1(Bna A01g0360D)in the restorer line materials,and got 6sterile single plants.It was found that the sterile phenotype was similar to the sterile line but the petals became smaller.It was verified by RNA-EMSA that the Bna.MORF1 protein directly acts on the m RNA of orf224,while the PPR protein encoded by the restorer gene does not directly act on orf224.It is speculated that the Bna MORF1 protein forms a protein complex with the Rfp protein to edit the sterility gene and cause infertility.The gene loses its function and fertility is restored.5.Multi-omics joint analysis to find differential genes related to CMS,using high-generation polima cytoplasmic male sterile near-isogenic line materials for sterile lines and reproductive lines,taking flower buds with a diameter of <1mm to detect proteome and metabolism Group,using laser microdissection capture microscope to cut tapetum cells of the same size flower buds for single-cell transcriptome sequencing;a total of 7598 proteins were identified in the protein group,among which 140 significantly different proteins were obtained(P-value<0.05,S/F>1.5);the metabolome detected a total of 699 metabolites,of which 37 were differential metabolites(|Fold change|?1 and |Fold change|?0.5),and single-cell transcriptome analysis found a total of 831 metabolites Differential gene(FDR<0.05).Finally,through the association analysis of transcriptome,proteome and metabolome,24 candidate genes were obtained.Using yeast two-hybrid technology,point-to-point verification of these 24 proteins was performed on the candidate genes screened by the association analysis.The result was RNA editing and respiratory electron transfer.The 13 proteins involved in chain,anther development,energy transport,tapetum development,and oxidative phosphorylation interact with orf224 protein and Rfp protein.