Selection And Genetic Engineering Improvement Of New Gerpmlasm Fae1 Mutant For High α-Linolenic Acid In Camelina Sativa L.

Camelina[Camelina sativa（L.）Crantz]is a new type of oilseed crop in the Brassicaceae family,which has excellent cultivation characteristics of high yield and stress resistance.Camelina seed oil is rich in essentialα-linolenic acid（18:3）,which is a functional plant oil for cerebrovascular diseases.In plant seeds,the two fatty acid desaturases,FAD2 and FAD3,convert oleic acid into linoleic acid and then convert linoleic acid intoα-linolenic acid by dehydrogenation,respectively.Oleic acid can also be catalyzed by fatty acid elongase 1（FAE1）to form very-long chain fatty acids that are harmful to human,and also shallows oleic acid substrates.In this study,the fae1 mutant obtained by gene editing was selected for the 18:3 content and the oil content of individual plants.Meanwhile,genetic engineering was used to improve the fatty acid components of Camelina to obtain high 18:3content material.The main results of this study are as follows:（1）In this study,the fae1 triple mutant of Camelina was selected by single plant selection and identification of progeny lines,and the high 18:3 and high oil content lines were selected,which had excellent agronomic characters.The relative content of 18:3 of the initial fae1 population was 45.4%,the oil content was 36.3%,the 18:3 grain content was205.6μg,and the average yield per plant was 2.5 g.In the first generation,100 lines with good agronomic characteristics were selected and 10 individual plants with an excellent18:3 content and oil content were selected.In the second generation,the 18:3 relative content of the lines No.3,No.26 and No.70 lines were over 47%,and the relative content of f 86-12α-linolenic acid was 50.6%.The relative oil content of f 37-9 was 48.1%,and the18:3 content of f 37-9 was 296.8μg.The 18:3 content of f 82-5 was as high as 1.8 g.The average yield per plant of these lines was also improved to 4.2 g.These results indicate that individual plant selection breeding is effective for comprehensive selection of superior gerpmlasm materials with highα-linolenic acid.（2）The AtFAD2NEW-BnaFAD3 co-expression vector constructed with the 2A peptide strategy was used to test the effect on seed fatty acids in Arabidopsis thaliana（L.）:In Arabidopsis Col-0 and fae1 mutants,the fatty acid components of the T₁and T₂positive seeds changed the same,and the relative content of T₁decreased,In the generation of T₂,the relative content of 18:3 of 10%lines increased by 4-12.9%,and the relative content of 18:3 of more than 90%lines decreased,suggesting that cosuppression of FAD3 occurred.Therefore,the vector was overexpressed in rdr6-11 mutant,and the relative content of 18:3 in generation of T₁and T₂was increased by 1.25 times,indicating that the vector was correctly constructed and FAD2 and FAD3 were effectively overexpressed,which also indicated that cosuppression did occur in transgenic lines in Col-0 background and the fae1 mutant background.When AtFAD2NEW-BnaFAD3 and ami RDR6 were transformed into fae1 mutant of Arabidopsis,the 18:3 relative content of T₂line was 13%higher than that of the control group,which reached 34%.These results indicated that ami RDR6 could relieve cosuppression of FAD3 in plants and further increase the relative content of 18:3.It was proved that the combination of double transformation AtFAD2NEW-BnaFAD3 and ami RDR6 inhibition could effectively increase 18:3 relative content.（3）Effects of FISET2 and FISET3 alone overexpression on fatty acids of Camelina seeds:After FISET2 transformed fae1 mutant Camelina,the 18:3 relative content of 40%of the 10 lines in the T₂generation increased,among which 18:3 relative content of line No.8increased by 9%to 50%compared with the control group,and the relative oil content of strain No.8 increased by 7%compared with the control group.FISET3 transformed into the fae1 mutant of Camelina.Among 18 lines of T₂generation,18:3 relative content of No.5 was increased by 2.5%compared with the control and 23.8%compared with the control,reaching 44.5%.These results indicated that overexpression of FISET2 and FISET3 could increase the 18:3 relative content of fae1 mutant Camelina,which could be used to create highα-linolenic acid camelina gerpmlasm.（4）Effects of combined expression of AtFAD2NEW,BnaFAD3 and FISET2 on seed fatty acids:Overexpression of AtFAD2NEW-BnaFAD3-FISET2 in Arabidopsis:Col-0 and fae1 mutants resulted in decreased relative content of 18:3and co-inhibition of FAD3 in T₁seeds.In the rdr6-11 mutant,the relative content of 18:3 increased by only 2.6%.The vector was overexpressed in the fae1 mutant Camelina linolenic acid,and the 18:3 relative content was increased by 3.3%-8.4%,but the 18:3 relative content was not significantly increased,and it was lower than the double gene expression of the AtFAD2NEW and BnaFAD3,suggesting that FISET2 was not suitable for co-expression with FAD2 and FAD3 to increaseα-linolenic acid.（5）Effects of combined expression of AtFAD2NEW,BnaFAD3 and FISET3 on seed fatty acids:Arabidopsis Col-0 and fae1 mutants were overexpressed,and the 18:3 relative content in T₁increased by 18%and 4-6%,respectively.In the context of rdr6-11 mutants,the18:3 relative content in 90%of T₁generation seed lines increased by 18.2%.The relative content of 18:3 of all lines in the generation of T₂increased significantly,and the highest was 44.9%,while the corresponding contents of 18:1 and 18:2 decreased.The results showed that the vector was correctly constructed and the gene could be effectively expressed.The addition of FISET3 indeed reduced the cosuppression frequency of FAD2 and FAD3.The result showed that the relative content of 18:3 in overexpressed lines of FAD2 and FAD3in the rdr6-11 was 0.6%higher than that in FAD2 and FAD3 tandem vectors.These results suggest that FISET3 in combination with FAD2 and FAD3 can enhanceα-linolenic acid.The tandem vector was transformed into the Camelina fae1 mutant,and the results showed that the relative content of 18:3 of T₂generation line increased by 2.5%,and the relative oil content increased by 23.8%to 44.5%compared with the control.To overcome the cosuppression in Camelina,artificial mi RNA was used to silencing RDR6（ami RDR6）.The tandem vector and ami RDR6 were transferred into the Arabidopsis fae1 mutant.The relative content of18:3 was increased in 20%T₂generation lines,and 18:3 relative content in No.18was 21%higher than that in the mutant,up to 42%,suggesting that ami RDR6 could release the cosuppression of FAD3 in plants and further increase the 18:3 relative content.However,the transgenic seeds of the double-transformed Camelina were not obtained due to the time.These results provided a helpful information for increasing 18:3 content in the fae1 mutant Camelina.