Construction And Functional Study Of BnaTT8 Mutants In Brassica Napus L.

Rapeseed is an important oil crop in our country.Many studies have shown that yellow-seeded rapeseed has many advantages over black-seed rape in terms of quality.However,the predominantly cultivated B.napus naturally lack yellow-seeded mutants.Presently,most yellow-seeded rapeseed in production were derived from distant hybridization.However,the generation of yellow seeded rapeseed varieties through the artificial synthesis method is not only time-consuming and labor-intensive but also causes extreme variation in seed color stability,which is not conducive to the breeding application of yellow seed traits.Researchers have carried out a large number of genetic and gene mapping studies on resynthesized yellow-seeded rapeseed.The results show that the inheritance of yellow-seed traits in these materials is very complicated,and the relevant genes have not yet been cloned.This greatly limits our understanding of the molecular mechanism regulating yellow-seed traits in B.napus.Findings from previous studies using Arabidopsis and B.rapa revealed that TT8 is an important gene involved in the formation of yellow of yellow-seed traits.In this study,we demonstrate the first application of CRISPR/Cas9 to target Bna TT8gene for the creation of yellow-seeded mutants in rapeseed;we studied the mechanisms involved in the regulation of the seeds oil and protein contents,as well as fatty acid composition and also conducted a transcriptomic and metabolomic analysis to explore the seed colour regulatory mechanisms.The research results are as follows:1.According to bioinformatics analysis and gene cloning,it is preliminarily determined that there are 3 homologous copies of Bna TT8 gene in the B.napus genome.Using q PCR technology to analyze the expression patterns of the Bna TT8gene in different tissues of B.napus transformation receptor J9707,we found that no transcript was identified for the Bna C09.TT8a,but various amounts of transcript were detected for Bna A09.TT8 and Bna C09.TT8b in all tissues,with the highest expression in seeds.Evidently,Bna A09.TT8 had a significantly higher expression level than Bna C09.TT8b in these tissues.By analyzing the expression level of both Bna A09.TT8and Bna C09.TT8b at different stages of seed coat development,revealed that their expression levelof exhibited a steady increase from 7 days after flowering（DAF）to a peak value at 21 DAF and then decrease accordingly at later stages.By analyzing the protein functional domain of TT8,it was further determined that the Bna TT8 gene has two functional copies in the B.napus J9707,Bna A09.TT8 and Bna C09.TT8b.2.The CRISPR/Cas9 vector used in editing the Bna TT8 gene was designed and constructed.The resulting construct was transformed into B.napus J9707,and 420 T₀generation transformed plants were generated.A total of 48 targeted mutants were identified through Sanger DNA sequencing of the PCR products of the target sites.After two generations of selfing and screening,9 homozygous mutants without T-DNA insertion were detected.Only the double mutants could produce yellow seeds,indicating that the two copies are functionally redundant.3.Using the vanillin and DMACA staining test to determine the dynamic variation of PA（proanthocyanidin）accumulation during seed development,we found that PA accumulation started at 21 DAF in the black seed coats of both WT and single mutants,and the accumulation increased steadily throughout the process of seeds development,except in the double-mutant seed which showed no obvious PA accumulation.4.Microscopic observation revealed that the accumulated PA were deposited in the endothelial cells of the WT and single-mutant seed coats.A comparative analysis of the seed coat thickness of mature seeds showed that the double mutant and the Bna A09.TT8 single mutant had a significant decrease in the thickness of the seed coat with respect to WT.5.We also measured the fatty acid（FA）composition,oil and protein contents of all double-mutant seeds of T₀,T₂ targeted mutations and WT.In comparison to the wild-type seeds,the oil and protein contents of the double mutant increased significantly,and the FA composition also changed significantly.The field test results showed that the main yield-related traits of the mutant materials did not change significantly compared with the WT,indicating that the mutated gene has a greater potential for application.High-throughput sequencing of the PCR products of the 26potential target sites from 30 T₀ gene-edited plants exhibited no off-target,indicating that the genome editing system used in this study has good specificity.6.The seed coats at 14 DAF and 35 DAF for comparison of the expression profiles between the Bna TT8 double mutants and corresponding WT,a total of 1,298differentially expressed genes between each double mutant and its corresponding WT,among which 145 DEGs were shared in common by the two stages.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis for these identified DEGs showed that the phenylpropanoid and flavonoid metabolic processes were significantly enriched among the down-regulated DEGs in mutants relative to WT.Metabolite profiling of the mature seeds of both the double mutant and wild-type was analysed,the results showed that the metabolites content in the flavonoid synthesis pathway of the double mutant was significantly reduced.7.The gene expression patterns of several important transcription factors and key enzymes involved in FA biosynthesis and accumulation in the mutant seeds and WT at different developmental stages were analyzed.The results showed that compared with WT,the expression levels of Bna FUS3 and Bna FAD2 was significantly up-regulated in the Bna TT8 mutant seeds at 14 DAF and 28 DAF,except for Bna LEC1 which was significantly up-regulated at 14 DAF.These findings revealed the significant roles of Bna TT8 gene in controlling fatty acids biosynthesis and accumulation.In summary,this present study successfully created a mutated Bna TT8 gene in Brassica napus and also confirm that it has important functions in regulating seed color,protein and oil content,and fatty acid composition.Furthermore,we also elucidate the regulatory mechanism of Bna TT8 in the regulation of flavonoids accumulation in the seed coat and the synthesis of seed fatty acids through the transcriptome and metabolome.This study lays a good material foundation for further research on the function of this gene and its regulatory molecular mechanism and also provides excellent germplasm resources for yellow seed breeding of Brassica napus.