Metabolic Engineering Of Plant Oils In Vegetative Tissues

For the last 100 years,the humanity economic society development are highly dependent on unsustainable fosil energy,which is declining in availability,increasing in price and is a major source of undesirable greenhouse gas emissions and environmental pollution,Because of over exploition and consumption.Plant oils have the potential to provide functionally equivalent,renewable and environmentally friendly replacements for these finite fossil-based raw materials,provided that their composition can be matched to end-use requirements.Triacylglycerols(TAGs)is main composition of plant oil,providing an important source of nutrition of human and animals,and applying in industry as margarine,salad oil,drying oil,biofuel and other chemicals.TAGs is limited to a few oil crop species,such as palm,soybean,rapeseed etc.Further,because of restrictions on arable land for oil crop growth,and the increasing demand for vegetable oil,the conventional oil crops are impracticable to produce biofuel in large scale.Plant oils enriched in omega-7(?-7)monounsaturated fatty acids,including palmitoleic acid(16:1?9)and cis-vaccenic acid(18:1?11),have nutraceutical and industrial value for polyethylene production and biofuels.Some studies showed that(?-7 have also ascribed a number of beneficial health properties,including promotion of cardiovascular fitness and reduction in LDL cholesterol levels.And biofuel produced by plant oils enriched in ?-7,have more advantages in enhancing cold flow properties,oxidative stability,NOx emission and ignition quality.However,existing oilseed crops accumulate only small amounts(<2%)of these novel fatty acids in their seed oils.A few of nonagronomic plants species can synthetize and accumulate much ?-7 in seeds,including seeds of Dolichandra unguis-cati,Kermadecia sinuata,and in some alages.Fortunately,C16:1?9 fatty acid's precursor-C16:0 fatty acids is maior protion of TAGs in most oil crops.As a result,metabolic engineering of ?-7 fatty acid production in existing oilseeds is the most promising approach for generating vegetable oils enriched in ?-7 fatty acids.Therefore,there is an urgent demand to establish sustainable technologies and develop new alternataive oil plant species,accumulating more TAGs enriched in ?-7 fatty acid and other quality fatty acids,to meet an expected future vegetable oil demand.In this study,high-biomass tobacco is employed as the model plant by systematic metabolic engineering,and developing a novel tobacco for accumulating higher oil,enriched in ?-7 fatty acid,in the vegetative tissues.The primary contents are as follows:1.VgDGAT1a gene was identified from Vernonia(Vernonia galamensis L.)to ectopic express in tobacco(Nicotiana tabacum L.)to enhance the oil content of vegetive issuees.The activity of VgDGATla enzyme was evaluated by N.benthamiana transient expression system,the result showed that expressing VgDGATla gene could increase the oil content in leaves of N.benthamiana,and there was not negative effect on the chlorophyll content.Then,VgDGATla gene was cloned into the binary overexpression vector,which was transformed into disk of tobacco by Agrobacterium tumefaciens-mediated method,and obtained transgenic plants overxepressing VgDGATla gene.The oil content of leaves and fatty acids composition were determined by gas chromatography.The results showed that oil content of transgenic tobaocco leaves enhanced 3.5-5.0 times compared to wild type,and the most is 9.2%(dry weight,DW).There was large change in composition of fatty acids,including C18:2 fatty acid increasing notablely,whereas C18:3 fatty acid reducing.And C20:0 fatty acid,no exsiting in bobacco leaves,was detected in transgenic plants.These demonstrated that the function of VgDGATla gene espressed in leaves was similar in seed.2.MucACP-?9D gene identified from Dolichandra unguis-cati was cloned into binary overexpression vector,which was transformed into discs of tobacco by Agrobacterium tumefaciens-mediated method,and obtained transgenic plants overexpressing MucACP-?9D gene.Gene expression was located in Chloroplast by western blot assay.Notablly,the composition of fatty acids in transgenic tobaocco leaves oil was different to wild type,including content of C16:0 saturated fatty acid decreasing 30-50%,the ?-7 of C16:1?9 and C18:1?11 increasing 16.4?20.4%?9.0%respectively.Statistical analysis showed that the content of ?-7 was strong negative correlation to the content of C16:0.The content of C18:2 and C18:3 polyunsaturated fatty acids in transgenic plants were reduced significantly,and statistical analysis showed that the content of two polyunsaturated fatty acids was negative correlation to the content of ?-7.All of these data showed that MucACP-A9D enzyme desaturated C16:0 to C16:1?9 with high acticity,and C16:1?9 was elongated to C18:1?11.At last,the content of C16:1?9 and C18:1?11 increased to 25.4?29.7%totally compared to little in wild type.3.Specific guide RNAs(gRNA)were designed to target AGPase and FatB gene,and most active gRNAs determined by in vitro experiments.The most active gRNAs were selected to contruct CRISPR/Cas9-AGP and CRISPR/Cas9-FatB binary vectors respectively.CRISPR/Cas9-AGP was transformed into discs of tobacco and D8 plant of expressing VgDGAT1a gene by Agrobacterium tumefaciens-mediated method,and obtained TO plants.Sequenceing results showed that mutant type and rate are deferent by the same CRISPR/Cas9-FatB binary vectors.But the change of carbohydrate of two plant type was similar,including reduction in starch and increment in total soluble sugar and reducing sugar.The oil content of mutant knockouted AGPase gene in wildtype had no obvious change compared to wildtype.The oil content of mutant knockouted AGPase gene in D8 plant overexpressing VgDGAT1a gene increased much more compared to wildtype.The results indicated that increasing more oil content need combine knockouting AGPase gene with overexpressing VgDGAT1a gene.Taken together,these results showed that valuable plant oil enriched in ?-7 fatty acid,could be increased considerably in plants vegetative tissues.The study results will provide new insights into understanding the mechanism of fatty acid and oil biosynthesis,based on CRISPR/Cas9 system in plant vegetative tissues,and also develop a new technology system for lipid metabolic engineering in plant vegetative organs as well as a production platform of biofuel oil using vegetative organs of non-food commercial crops.