Lipid Dynamic Accumulation And Function Analysis Of Key Genes In Developing Fruit Of Sea Buckthorn(Hippophae Rhamnoides L.)

Sea buckthorn oil is rich in a variety of biological active substances,which are widely used in nutrition,medicine,cosmetics,biofuels,light industry and other fields.Moreover,as a drought-tolerant woody plant,seabuckthorn can grow on the marginal land such as sandy land,barren mountain and saline-alkali land,so as to avoid competing with field oil crops for land.Therefore,sea buckthorn is an ideal oil supply object.The seed of sea buckthorn is rich in human essential fatty acid,α-Linolenic acid(C18:3N3).Most studies on sea buckthorn focus on fatty acid composition and fatty acid determination methods,but few studies have focused on the synthesis mechanism of sea buckthorn.Triacylglycerol(TAG)is the main form of oil found in plants and is stored in oil bodies.It can be used in food,biofuel,light industry and other fields.It can also be used as raw material for biodiesel production and promote the development of new renewable energy.Although there are some studies on the synthesis of TAG in oilseed crops such as rapeseed,there are few studies on how the fatty acids in sea buckthorn synthesize TAG and the enzymes and key genes affecting the synthesis of TAG in sea buckthorn.In order to further develop and utilize sea-buckthorn oil,it is essential to better understand the lipid metabolism in sea-buckthorn at the molecular level.The main research methods and results are as follows:(1)The fatty acid components of Hippophae rhamnoides ‘Mongolia’(XY)and H.rhamnoides ‘Sinensis’(FN)sea buckthorn seeds and pulp at three different development stages were determined by gas chromatography-mass spectrometry.Saturated fatty acid(SFA)was mainly accumulated in the pulp of sea-buckthorn.In the three stages(T1～T3),the saturated fatty acids in XY pulp accounted for 64%,67% and 57% of all fatty acids,and those in FN pulp were 64%,60% and 65%,respectively.The relative content of saturated fatty acids in the pulp of sea buckthorn remained stable in the T1～T3 period,and the change was not significant,indicating that the desaturation effect of saturated fatty acids was not significant,leading to the low content of unsaturated fatty acids in XY and FN pulp.Instead,sea buckthorn seed mainly accumulated in the unsaturated fatty acids(UFA).During the maturation period(T3)of seabuckthorn seeds,unsaturated fatty acids in XY and FN accounted for 79% and 78%of all fatty acids.Mainly for polyunsaturated fatty acids(PUFA).The PUFA/ UFA of the two cultivars were similar,62% in XY seeds and 63% in FN seeds.The polyunsaturated fatty acid with the highest content in XY seeds is α-linolenic acid,while the polyunsaturated fatty acid with the highest content in FN seeds is Linoleic acid(C18:2).(2)Alpha-linolenic acid is an essential fatty acid,which plays an important role in brain development,cardiovascular health,and the treatment of inflammation.Sea buckthorn seeds are rich in α-linolenic acid.The highest content of fatty acids in XY seeds is α-linolenic acid,followed by linoleic acid.The content of α-linolenic acid in FN seeds was lower than linoleic acid.The results showed that the expression levels of gene FAD2 and gene FAD3 in XY were3.83 times and 13.63 times higher than that of FN in the semi-mature stage(T2).The expression levels of gene FAD7 in XY in the immature stage(T1)and T2 stage were significantly higher than that in FN,which were 2.09 times and 1.72 times respectively.It is consistent with the trend of linoleic acid and α-linolenic acid content.On the contrary,the expressions of gene LOX3 in FN were higher than XY in T2,which were 8.02 times.The high accumulation of α-linolenic acid in sea buckthorn seeds was due to the synergistic effect of multiple genes.High expression of gene FAD2,FAD3,FAD7 and low expression of gene LOX3 contribute to the accumulation of α-linolenic acid.(3)Fatty acids and glycerol were catalyzed by a variety of enzymes to produce TAG on the endoplasmic reticulum.TAG is the main form of oil found in plants and is stored in oil bodies.Through lipidomics analysis,it was found that the accumulation pattern of TAG in XY and FN seeds was different.In XY seeds,TAG content increased gradually during seed development and reached a peak in T3.However,in FN,TAG content was close to the peak in T2 period,and TAG accumulation was slow after T2 period.There was no significant difference in TAG content between T3 and T2.Transcriptome was used to analyze the differences in gene expression trends between two seabuckthorn varieties.Two genes that play an important role in TAG synthesis were identified and a gene regulatory network was constructed.In sea buckthorn seeds,Gene DGAT1-A(Gglean024650)played a role in the final step of Kennedy pathway,promoting the acylation of diacylglycerol(DAG)to form TAG.The expression trend of gene DGAT1-A in two sea buckthorn cultivar s was consistent with that of TAG.The gene PDAT1-A(Gglean008591)can acylation DAG to form TAG through an acyl-coenzyme independent pathway.Through VIGS,q RT-PCR and lipid analysis,it was found that gene DGAT1-A and gene PDAT1-A had similar functions in sea buckthorn seeds.All can synthesize TAG with DAG as substrate.These two genes together contribute to the steady increase and accumulation of TAG in sea buckthorn seeds.At the same time,it was found that the silencing of PDAT1-A gene could affect the wax synthesis of sea buckthorn seeds.