Development Of Novel Germplasms With Altered Fatty Acid Profiles Through Genome Editing Technology In Brassica Napus

Rapeseed is one of the largest oil crops after oil palm and soybean in the world.The quality of rapeseed oil was largely determined by the fatty acid composition and the proportion of individual fatty acids.Oleic acid benefits in reducing cholesterol,suppressing tumor formation and preventing arteriosclerosis inflammatory diseases.vegetable oil with high stearic acid content can not only save hydrogenation costs but also avoid the production of trans fatty acids,and has advantages such as no effect in cholesterol concentration.Low content of linolenic acid is essential for the production of healthy food due to it can prevent the oxidation and rancidity in rapeseed oil.Therefore,there is much need of genetic improvement of rapeseed for creating novel materials with specific fatty acid content according to the purpose.In the present study,the mutations were introduced into five essential genes,BnaFAD2,BnaSAD,BnaFAD3,BnaFAE1 and BnaFAE1.Like,which were responsible for the regulation of fatty acid biosynthesis,via CRISPR/Cas9 genome editing technology.A systematic analysis was conducted to quntify the changes of the fatty acid contents in different types of mutants.The main results are as follows:1.Four copies of the BnaFAD2 gene were cloned from the pure lineJia9707 in B.napus and the editing vector named pTG8 was constructed.Finally,different types of mutants were identified in three copies of BnaFAD2.A5,BnaFAD2.Al and BnaFAD2.C5.Statistical analysis of the mutation types suggested that most of the mutated types introduced by the CRISPR/Cas9 genome editing system were small insertion and deletion in Brassica napus.Two mutants located in different locus were used to monitor the heritable transmission rate on the T1 generation and the results demonstrated that the transmission rate of mutations for both loci were consistent with a single Mendelian inheritance pattern.Furthermore,off-target analysis had shown that there were no off-target phenomena was observed in Jia9707.2.Ten lines with mutations at BnaFAD2.A5 were obtained.The content of oleic acid for all mutant lines at BnaFAD2.A5 was significantly increased compared with wild type.The oleic acid content ranged from 73.1 to 82.3%,while the content of linolein and linolenic together decreased,ranging from 18.4%to 11.1%.A 80bp-deleted line,PTG8-59-18-40-12(a5a5),showed the highest oleic acid content(82.3%)among all the mutants.3.Furthermore,we obtained two homozygous lines with constant mutations at BnaFAD2.C5 loci.The oleic acid content of both lines increased significantly in the seeds and up to 73%and 74%,respectively,which showed weaker effect than the mutant of BnaFAD2.A5.A set of three mutant plants at BnaFAD2.A5,BnaFAD2.C5 and heterozygous mutant of double mutants at two loci,respectively,were used for single seed analysis to look into the segregations of oleic acid content.The results of single seed analysis for fatty acid composition showed that oleic acid content could be further increased by combining these two mutation alleles.4.The AtSSI2 gene of Arabidopsis was used as a reference to identify four members of the BnaSAD gene in the Brassica napus genome.Conserved domain analysis revealed that all four genes have the conserved domain of acyl-[acyl carrier protein]desaturase,and the amino acid sequence is highly similar to with the ones of their Arabidopsis homologue.RNA-seq data showed that different copies of the BnaSAD gene exibited a unique spatiotemporal expression pattern in B.napus.5.Two editing vectors,named pSAD1 and pSAD2,were prepared for targeting all four genes of BnaSAD.Different types of mutant lines were identified including single locus mutants at the four genes and double mutants of BnaSAD.A3 and BnaSAD.C3,BnaSAD.A3 and BnaSAD.A5 and BnaSAD.C3 and BnaSAD.C4.The content of stearic acid for all mutants increased significantly compared with wild type.Comparative analysis of different types of mutants showed that BnaSAD.C3 had the strongest effect on increasing stearic acid content,which was up to 8.8%;the effect of BnaSAD.A3 loci was after BnaSAD.C3 loci,which was up to 8.4%.Furthermore,BnaSAD.A3 and BnaSAD.C3 had cumulative effects in stearic acid content,which could increase the stearic acid content up to 14.5%.6.Analysis of the different types of mutants suggested that BnaSAD.A 5 gene mutation can cause obvious leaf yellowing at the seedling stage.Further analysis demonstrated that combination of different mutation alleles at two genes,such as BnaSAD.A3 and BnaSAD.A5,had cumulative effects for leaf yellowing and can lead to the degree of yellowing more serious.7.Transcriptome data analysis indicated that the six copies of the BnaFAD3 gene had different spatiotemporal expression patterns,and the expression reached the maximum value at 34 days after flowering.The knockout vector named pBnaFAD3 was constructed to target the BnaFAD3 genes.A mutant was identified on the 5’-UTR of the BnaC03g16520D copy.The mutation leads to the increased oleic acid content up to 70.5%,while the linolenic acid content decreased to 4.5%in the seeds.8.Transcriptome data analysis for the expression of the BnaFAE1 gene revealed that the gene was expressed during the different development stages of seeds in the low erucic acid material.However,the expression level was very low and the expression level increased dramatically to the peak at 34 days after flowering.At the same time,BnaFAE1-Like.A3 and BnaFAE1-Like.C3 gene were shown highly homologous with the BnaFAE1 genes in the Brassica napus genome.The analysis of the conserved domain and protein localization predicted that these two genes may have similar functions with BnaFAE1 genes.Two editing vectors,pBnaFAEl and pBnaFAE1.Like,were constructed to target BnaFAEl and BnaFAE1-Like.A3 genes,respectively.The fatty acid analysis showed that the mutation of the BnaFAE1 gene caused the content of stearic acid increasing to 3.2%,and the mutation in the BnaFAE1-Like.A3 gene lead to increase stearic acid to 4.2%in rapeseed.