Study On The Restore Mechanism Of BnaMs3 In The Genic Male Sterile System 7365 In Brassica Napus

The recessive genic male sterile line 7365 A of Brassica napus L.is a complex allelic inheritance model controlled by two loci(Bna Ms3/Bnams3,Bnams4a/Bnams4b/Bnams4c),of which Bna Ms3 and Bnams4 a are restorer genes,and Bnams4 b is a sterile gene.Bnams4 b causes degradation of the stamens of canola and leads not to produce mature pollen.Bna Ms3 can rescue this sterile phenotype and make the plants have pollens,and this genetic pattern is also applicable in Arabidopsis by transferring two genes.Previous studies had suggested that Bnams4 a regulates the lower expression of Bnams4 b at the transcriptional level by DNA methylation,and Bna Ms3 interacts with Bnams4 b at the protein level.Evolutionary analysis and genetic transformation experiments of Bna Ms3 showed that Bna Ms3 is a functionally acquired variant gene,and that the functionally obtained mutation site may exist within the six functional amino acid residues of TPR and Hop.In this study,CRISPR/Cas9 technology was used to edit the gene A41 encoding the interaction protein with Bna Ms3 and Bnams4 b,and the mechanism of action of Bna Ms3 was initially investigated.In the meantime,site-directed mutagenesis for the six predictive functional variants of Bna Ms3 was combined with transfer of Bnams4 b sterile material,respectively,to explore the functional variant amino acid sites and functional domains of Bnams3.The results were listed as following: 1 A41 gene editingThe gene A41 encodes a protein that interacts with both Bna Ms3 and Bnams4 b screened in a yeast two-hybrid assay.A41 has been reported to function as a protease inhibitor,and it is speculated that it participates in the process of Bna Ms3 restoring Bnams4 b.By editing of the wild-type Arabidopsis thaliana A41 gene of the CRISPR,homozygous A41-edited and non-transgenic plants were obtained.By crossing into the plants where both Bna Ms3 and Bnams4 b are present,the expected altered phenotype for fertility was not obtained.It was speculated that A41 might not be involved in the Bna Ms3 recovery of Bnams4 b.2 Exploration of Bna Ms3 Functional Variable Amino Acid SitesFor the predicted six Bna Ms3 functional variant amino acid sites,six site-directed mutagenesis expression vectors for Bna Ms3 were constructed to transform wild-type Arabidopsis thaliana and crossed with Bnams4b-transgenic Arabidopsis thaliana,respectively.Phenotypic observation and acetate carmine staining of offspring showed that Bna Ms3 lost its restoring function after mutation of 321 amino acids in Bna Ms3,presumably altering the protein structure of Bna Ms3 after a single amino acid mutation,thereby affecting the function of Bna Ms3.3 Bna Ms3 regulates the expression of Bnams4 b at the transcriptional levelSite-directed mutagenesis showed that an amino acid mutation in the TPR domain would cause Bna Ms3 to lose its recovery function.TPR protein has important functions in plastid development and gene expression and participates in plastid RNA cleavage.q RT-PCR results showed that Bna Ms3 reduced the expression of Bnams4 b.It is speculated that Bna Ms3 regulates the expression of Bnams4 b at the transcriptional level,and Bna Ms3 as a TPR protein restores the plasminic RNA-sparing defect,thus relieving Bnams4 b of the destruction of the plastid and restoring the fertility.