Improvment Of Oil Quality In Brassica Napus USING CRISPR/CAS9 Gene Editing Technology

Rapeseed is an important oil crop in China,and the quality of edible oil directly affects human health,so rapeseed quality improvement breeding is particularly important.The main component of oil in rapeseed is triglyceride,which consists of fatty acid chain and glycerol skeleton,of which fatty acid is the variable part.The composition of fatty acid and the corresponding ratio determine the quality of rapeseed oil,and breeding rapeseed varieties with high oleic acid,low linolenic acid and low erucic acid is an important direction for rapeseed breeding.By regulating the expression of specific genes,the composition of fatty acids in rapeseed oil can be precisely improved to meet the demand for oil in food processing,which is conducive to improving the economic benefits and comprehensive utilization value of rapeseed.Fatty acid desaturase 2（FAD2）,fatty acid desaturase 7（FAD7）and fatty acid elongase1（FAE1）are three key enzymes that regulate the synthesis of oleic acid,linolenic acid and erucic acid,respectively.In this study,Westar,a kale type oilseed rape,was used as the recipient material to knock out the genes encoding the above three enzymes using CRISPR/Cas9 gene editing technology,while the Bnafad2,Bnafad7 and Bnafae1 genes were polymerized by hybridization,expecting to obtain strains with increased oleic acid（C18:1）content and reduced linolenic acid（C18:3）and erucic acid（C22:1）content strains with reduced oleic acid（C18:1）and linolenic acid（C18:3）and erucic acid（C22:1）contents.The main results were as follows:1.Two copies of FAD2 were present in the oilseed rape genome,located on chromosomes A05 and C05,respectively.One FAD2 gene mutant was obtained in T₀generation.3 of 16 T₁ generation monocultures were edited,and the oleic acid content was64.77%,60.68%and 61.25%,respectively,all higher than the wild type（60.63%±1.34%）.42 monocultures were tested in T₂ generation,and 35 were edited.3 double gene pure mutants were selected and tested,and the average content of oleic acid was 60.89%±0.36%,which was highly significant from the wild type（55.28%±0.37%）.2.There are two copies of FAD7 in the oilseed rape genome,located on chromosomes A03 and C03.Two FAD7 mutants were obtained in the T₀ generation.The average linolenic acid content was significantly lower（5.71%±0.38%）than that of the wild type（7.23%±0.25%）in three selected double pure mutants.90 single plants were tested in the T₂generation and 78 were edited.Three double gene pure mutants were selected for testing and the mean linolenic acid content（8.53%±0.23%）was significantly lower than that of the wild type（10.20%±0.35%）.3.One FAE1 mutant was obtained in the T₀ generation.45 single plants were tested in the T₁ generation,43 were edited and three double pure mutants were selected for testing,with an average erucic acid content（0.30%±0.04%）compared to the wild type（0.35%±0.11%）.All were edited and three double pure mutants were selected and tested,with a lower mean erucic acid content（0.16%±0.00%）than the wild type（0.29%±0.03%）.4.T₂ generation double gene pure mutant plants were selected for crossing,and the Bnafad2 mutant was used as the parent to cross with the Bnafad7 mutant to obtain F₁generation bagged self.In the F₂ monoculture population,two convergent plants with Bna C05.fad2 pure,Bna A05.fad2,Bna A03.fad7 and Bna C03.fad7 heterozygous and one convergent plant with Bna C05.fad2 double allele,Bna A05.fad2,Bna A03.fad7 and Bna C3.fad7 heterozygous were screened and no plants with pure genotypes for all four genes were obtained.The Bnafad7 mutant was used as the parent to cross with the Bnafad2mutant and obtain the F₂ population,in which five heterozygous genotypic plants with aggregated Bna A05.fad2,Bna C05.fad2,Bna A03.fad7 and Bna C03.fad7 were obtained.The Bnafad2 mutant was used as the parent to cross with the Bnafae1 mutant,and one aggregated plant with Bna C03.fad1 pure and Bna C05.fad2 heterozygous and two aggregated plants with Bna C03.fad1 chimeric and Bna C05.fad2 heterozygous were screened in the F₂ population.The Bnafad7 mutant was used as the parent to cross with the Bnafae1 mutant,and one polymeric plant with Bna A03.fad7 pure and Bna C03.fad7 and Bna C03.fae1 heterozygous was screened in the F₂ population,but no plant with Bna A08.FAE1 mutation was obtained.Subsequent screening for pure mutants of each gene will be continued in the self-progeny of the above single plants and fatty acid content will be examined.In this thesis,mutants of the Bna FAD2,Bna FAE1 and Bna FAD7 genes were obtained using CRISPR/Cas9 gene editing technology,providing a material basis for the subsequent introduction of mutant genes into superior varieties.