CRISPR-Cas9 System Mediated For Genome Targeted Editing And Functional Analysis Of FAD2 In Flax(Linum Ustitatissimum L.)

Linseed is one of the world's oil seed crops,and it's a kind of crop with high commercial value.Flax has been cultivated for a long tim.In terms of its yield,China is now the second largest country in the world.The unsaturated fatty acids in flax contain unsaturated double bonds.The more unsaturated double bonds there are,the worse the stability will be,and the oil more easily oxidize will be,which is not conducive to transportation and storage.Therefore,it is essential to improve the stability of oil.We focused on the key enzyme which control the oleic acid dehydrogenation into linoleic acid in the fatty acid metabolic pathway--?12-fatty acid desaturase(FAD2).In this experiment,CRISPR technology was used to edit the FAD2 gene of flax "LongYa No.10" for oil,and the genetic transformation was carried out by tissue culture method and pollen tube channel method respectively.By controlling the synthesis of FAD2 enzyme,the metabolic pathway of fatty acids is controlled and the fatty acid composition is improved.We tested the composition and content of fatty acids in 200 flax seeds from 43 countries.Furthermore,genome-wide association analysis was performed on multiple SNP sites obtained from whole-genome sequencing of these 200 materials,and the genetic diversity of flax was studied using molecular markers to discover candidate genes related to the desaturation of flax fatty acids.The main research results are as follows:1.Applied the method of Golden Gate cloning to construct the gene editing vector for the FAD2 gene of �LongYa No.10�,named pYLCRISPR/cas9-FAD2.Afterwards,introduced the vector into Agrobacterium GV3101.2.Genetic transformation.122 strains of Hygromycin resistant callus were obtained by tissue culture,and 83 of them were positive callus that could be amplified by PCR with Hygromycin resistant gene and two target fragments.According to the sequencing results,compared with the wild type,1bp deletion occurred in the target sites of No.FAD2B-A5;1bp insertion occured in the target site of No.FAD2B-5;1bpreplaced occurred before the target sites of No.FAD2B-B4 and FAD2B-14.We successfully edited the FAD2 gene of �LongYa No.10�.3.A total of 15,000 flowers were transformed by pollen-tube pathway method,and more than 70,000 seeds were harvested.25 strains were screened with Hygromycin resistance.4.The contents of five different fatty acid components in 200 flax germplasm were determined by GC/MS.SPSS 2.0 software was used to analyze the contents of total lipids and fatty acids.The main contents of total lipids,palmitic acid,stearic acid,oleic acid,linoleic acid and linolenic acid were: 8.25-21.25mg/g,1.03-2.03mg/g,0.7-1.3mg/g,3.03-4.78mg/g,1.67-3.57mg/g,5.23-8.75mg/g respectively.In combination with GWAS,a total of 8 candidate genes related to oleic acid metabolism and 2 candidate genes related to linoleic acid metabolism were discovered.