Functional Characterization Of BnaEOD3/CYP78A6 In Brassica Napus By Using CRISPR/CAS9 System

Rapeseed?Brassica napus L?,is the second largest oil crop after soybean.B.napus?AACC,2n=4x=38?belongs to Brassicaceae family and has gained tetraploid genome due to natural hybridization between B.rapa?AA,2n=20?and B.oleracea?CC,2n=18?during evolution.EOD3 encodes a member of CYP78A cytochrome P450 monooxygenase protein family which mainly involved in seed growth.Seed size is an important agronomic trait affecting yield of the crop.Small-seeded plants produce large number of seeds due to its superior colonization ability.Comparatively,little research known about the genetic and molecular mechanisms that control final seed size in rapeseed.CRISPR/Cas9 has been widely used for improving various agronomic trait in different crop species,to enhance the yield and quality of crops and capable against different diseases.Seed size and number are central to the evolutionary fitness of plants and are also crucial for the seed production of crops.Here,we report the gene cloning,expression analysis,and functional characterization of the EOD3/CYP78A6 gene in rapeseed.In the present study,we focused on four homologs of BnaEOD3 gene?BnaA04.EOD3,BnaA05.EOD3,Bna C04EOD3a and Bna C04.EOD3b?in B.napus,which exhibited a steady higher expression during seed development with differential expression among copies.In B.napus no natural or induced mutations of BnaEOD3 gene have been studied up to now,mainly due to its allotetraploid nature and multiple gene copies.The CRISPR/Cas9 system was utilized to induce knockout mutations in BnaEOD3.The CRISPR/Cas9-induced mutations in four homologs of BnaEOD3 gene were confirmed by PCR and Hi-TOM sequencing method.The Mutations frequency were observed up to 83-90%of BnaEOD3 from T₀to T₂generation.These mutations were stably transmitted to T₁and T₂generations and a large collection of homozygous mutants with combine loss-of-function alleles across four BnaEOD3 copies were created for phenotyping.The phenotypes of BnaEOD3 mutant lines showed that silique length,seed size and thousand seed weight?TSW?decreased while seed number and seed weight per silique and per plant increased.Consequently,the seed weight per plant in the quadrable mutants increased by13.9%on average compared with that of wild-type?WT?,indicating that these BnaEOD3copies have redundant functions in seed development in rapeseed.The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes.Furthermore,Bna C04EOD3a and BnaA05.EOD3 are most important homologs than other two copies of BnaEOD3 gene.Therefore,all knockout lines of four homologs of BnaEOD3 gene showed smaller seed size,short silique and more seed number.The cytological study also showed that mutants of BnaEOD3 developed small cotyledon as a result of the reduced cotyledon cell expansion and proliferation.The magnitude of the changes of mutant and wild type cotyledons closely parallels the variations in the areas of cotyledon cells.Comparatively,the homozygous mutant of BnaEOD3 had approximately?0.90 times fewer cells than that of wild type?0.68/0.75=0.90?.Collectively,our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development,but also provided valuable resources for rapeseed breeding programs.we searched for the putative off-target site in the B.napus genome with high homology to the four sg RNA by using CRISPR-P program.Total 47-potential off-target sites were found for S1-S4 from many T₀mutated plants,indicating no mutations according to Hi-TOM of the PCR products.Therefore,the chances of off-target effect are very low when the specificity of sg RNA is considered well according to the genome sequence.In the current study,we analysed the editing efficiency of each sg RNA?S1-S4?which target the four homologs of BnaEOD3 gene.These sg RNAs targeting the same gene showed dramatically capricious genome-editing efficiencies,such as 44.90%in S1,45.60%in S4,0%in S2 and 20.00%in S3.The sg RNA efficiency depends on its promoters in B.napus.S1 and S4 are driven by p At U3d and p At U6-29,while S2 and S3driven by p At U3b and p At U6-1,indicating that p At U3d and p At U6-29 showed better expression of sg RNA to an induced efficient mutation in the target sequence.Furthermore,the expression analysis of each copy of BnaEOD3 were analysed through q RT-PCR and identified the expression pattern of each copy of BnaEOD3 among different tissues.The four homologs of BnaEOD3 showed high expression in 7 DAF seed and 14 DAF silique among all tissues.The two copies Bna C04.EOD3a and BnaA05.EOD3 had the highest expression level than other two copies.We also further characterized the expression pattern of each copy of BnaEOD3 in different stages of seeds:7,14,21,28,and 35 days after flowering?DAF?.The expression of all four copies showed a steady increase from 7 DAF to the highest value at 21 DAF and then decreased at 28 and 35 DAF stages.Comparatively,we suggested that these four homologs of BnaEOD3 functionally identified that play a key role in seed size and silique length.can better be colonized and increase the yield production of B.napus in the global market.Consequently,the transgenic lines of BnEOD3 developed in the current study might be helpful in future for tremendous starting for high-yield breeding in canola.