Study On Modification Of Fatty Acid Composition In Camelina Sativa By Artificial Microrna Interference Technology

As the fossil fuels running out,it is imperative to looking for renewable energy,and sustain human existence as well as social sustainable development.The development of bio-fuels can effectively deal with a series of major issues such as energy,environment,climate change and so on.Screening excellent raw material is the key factor to accelerate the development of biofuel industry.Camelina sativa considered as one kind of low-input crop,is a promising alternative oilseed crop in many regions,such as food,feed and industrial applications.However,the fatty acid(FA)composition of camelina oil does not comply with the characteristics of an ideal fuel,and it is necessary to improve the content of oleic acid through biological means,during which FA profile was employed to select feedstocks high in monounsaturated FAs.Fatty acid dehydrogenase(FAD2)and fatty acid chain elongase(FAE1)are key enzymes for the synthesis of linolenic acid and eicosenoic acid,respectively.In this study,we describe an artificial microRNA approach to reduce the undesirable fatty acids in camelina.The genetic expression of FAD2 and FAE1 were suppressed by expression vector pBinGlyRed2-CsFADEamiR in order to improve the fatty acid composition of camelina oil.This technique also used for the transformation of camelina to be better used in various fields of food ingredients.The results of this study are showed as follows:Cas-miR159a was cloned from roots,stems,leaves and seeds(DAF20)of camelina through stem-loop primers designed.Quantitative Real-time PCR(RT-qPCR)analysis showed that the hightest expression of Cas-miRl 59a occured in the seeds(8.1),while the lowest one in root(1.0).The pBinGlyRed2-CsFADEamiR interference vectors was successfully constructed when CsFADEamiR was inserted into pBinGlyRed2.The screening marker of plant expression vector is kanamycin and red fluorescent protein(DsRed2).Agrobacterium tumefaciens was used as mediated strains.At early and bloom flowering stage,the expression vector plasmid was transformed into Camelina sativa named as Suneson through the vacuum infiltration procedure.We had successfully obtained 20 T1 transgenic seeds by the green LED light screening.GC analysis indicated that the content of fatty acids in transgenic seeds had been changed.The expression of FAD2 and FAE1 gene in transgenic lines was effectively inhibited.The best trandgenic line was T2-9.The relative content of oleic acid(18:1)increased from 15%to 32%.The relative content of polyunsaturated fat(18:2,18:3)decreased from 57%to 38%,while that of the long chain fatty acids(20:1)decreased from 11%to 3%.