Functional Validation Of Candidate Genes BnaCOL9 And BnaMYB109 At Flowering Time In Brassica Napus L.

As an important oil crop in China,rapeseed was widely cultivated in the Yangtze River basin.Flowering time is a very important agronomic trait,and timely flowering can not only solve the stubble contradiction with rice,but also avoid yield reduction and pest and disease erosion caused by weather factors.At the moment,the genetic regulatory network of flowering time in rapeseed has not been in-depth research,and further research into the genetic regulatory network of flowering time in rapeseed is vital.We incorporated the results of previous Genome-wide association study（GWAS）of flowering time in rapeseed and projected 20 candidate genes that may impact flowering time in this study.The previously identified 20 genes in rapeseed were targeted for mutation using CRISPR/Cas9 technology,and eventually mutations in BnaCOL9 and BnaMYB109 genes were discovered to produce flowering time alteration.The molecular mechanism of the BnaCOL9 and BnaMYB109 genes that regulate flowering in kale type oilseed rape was preliminarily resolved on this basis.The main findings were as follows:1.Through bioinformatic analysis and gene cloning,we identified four homologous copies of BnaCOL9 in rapeseed.Expression pattern analysis revealed that BnaCOL9 was mainly expressed in rapeseed leaves and flowers,and this result was verified by constructing the promoter vector BnaCOL9.A05 GUS and staining analysis after rapeseed transformation.Subcellular localization results of BnaCOL9 showed that its protein was localized in the nucleus.2.A CRISPR/Cas9 vector targeting BnaCOL9 was constructed to transform rapeseed Westar and 40 T₀ generation regenerated plants were obtained,38 of which were positive monocots.Editorial detection using Hi-TOM high-throughput sequencing technology revealed that the mutation types were mostly heterozygous mutations.The heterozygous mutant strains were examined phenotypically after bagging and self-crossing.The results showed that the BnaCOL9 mutation resulted in significantly earlier flowering compared to the wild type.To verify that BnaCOL9 was involved in regulating flowering time in rapeseed,we constructed a BnaA05.COL9overexpression vector and transformed rapeseed Westar.The results showed that BnaA05.COL9 overexpression resulted in a significant delay in flowering time,indicating that BnaCOL9 is indeed involved in the flowering process of rapeseed.3.Through yeast point-to-point experiments,we found that BnaCOL9 can interact with BnaRCD1,and this result was verified by luciferase complementation experiments.BnaRCD1 was edited using CRISPR/Cas9 technology to create mutants of BnaRCD1.Examination of the phenotypes of the mutants revealed that mutations in BnaRCD1also resulted in earlier flowering time in rapeseed.4.By bioinformatics analysis and gene cloning,we identified two copies of BnaMYB109 in rapeseed.Expression pattern analysis revealed that BnaMYB109 was expressed in all tissues,and subcellular localization results showed that BnaMYB109was localized in the nucleus.5 A CRSPR/Cas9 knockout vector of BnaMYB109 was constructed.It was also transformed into rapeseed Westar and 12 T₀ generation monocultures were obtained,10of which were positive.Editorial assays and phenotypic examination of the progeny showed that the BnaMYB109 mutation caused delayed flowering time in rapeseed.6.To explore the gene pathways affected by the BnaMYB109 mutation,we performed transcriptome sequencing of the mutant and wild type.Data analysis revealed a total of 9576 differentially expressed genes,of which 5174 were up-regulated and 4582 were down-regulated.KEGG enrichment analysis of the down-regulated genes revealed that the plant circadian pathway was significantly enriched,with the most significant down-regulated expression of the flowering gene BnaFT.Based on the above results,we speculate that BnaMYB109 may regulate the flowering period of kale type oilseed rape by affecting the expression of BnaFT genes.