Study On The Factors Affecting The Accumulation Of Very Long-chain Fatty Acids In Cruciferae Oil Plant Seeds

Very long-chain fatty acids(VLCFA,?20 carbons in length)are mainly derived from traditional rapeseed.The VLCFA has numerous industrial applications in chemical engineering,energy as well as food and drug manufacturing.Thus it is of great theoretical and practical significance to understand the biosynthesis and accumulation of VLCFAs in seeds.The seeds of Arabidopsis thaliana and Camelina sativa account 20%of VLCFA,making them ideal systems to study the mechanism of VLCFA biosynthesis and accumulation in oil plants.In order to accelerate the research process and improve the reliability of phenotypic identification,this study first optimized the quantification method of fatty acid composition and oil content in seeds.On this basis,this study was designed to discern the mechanisms of synthesis and accumulation of VLCFA in seeds of A.thaliana,C.sativa and Brassica napus were investigated through regulating the content of substrate,the abundance of fatty acid elongase(FAE)and pivotal acyltransferases.The main results are as follow:(1)Based on the optimizated conditions of seed methyl esterification,a method of single Arabidopsis seed mini-transmethylation(SAST)has been established to identify the fatty acid composition and oil content of single T₁ seed directly by identifying transgenic seeds with visible fluorescence.The direct transmethylation method was first optimized by employing multiple seeds and recommended conditions were incubated at 85°C for 2 hours in a transmethylation solvent(5%H₂SO4 in methanol with 30%toluene cosolvent).In the SAST system,200?l transmethylation solvent was added into a 1.5 m L GC sample vial.The standard reaction conditions and fatty acid methyl ester microextraction method were used and detected in a small via insert.Our results indicate that fatty acid composition of T₁ seeds can be detected by single seed because of their minor differences in fatty acid composition.However,oil content per seed varied substantially,and hence for phenotyping oil content of T₁ seeds,pooling five or ten seeds is recommended.The suitability of partial seed analysis of camelina was also investigated;however,variation in the composition of different seed tissues limits this approach.(2)FAE1 co-suppression is a main bottleneck to improve the VLCFA.About 90%transgenic lines were suppressed by expressing FAE1 in Arabidopsis wild type.It was found that Bn FAE1 from B.napus and Tm FAE from Tropaulum majus also causes co-suppression with different frequencies when transformed in Arabidopsis wild type,and a similar phenomenon was observed when FAE1 from A.thaliana transformed in camelina.Moreover,it was elucidated that co-suppression frequency was positively correlated with the sequence similarity index of homologous genes between transformed and host.Additionally,this co-suppression was effectively released when it was expressed in the rdr6-11 mutant host.(3)The expression level of FAE1 is the limiting factor in the accumulation of VLCFA in Arabidopsis seeds,but not the substrate's abundance(oleic acid,18:1).VLCFAs could not be improved by increasing the proportions of 18:1 from the high 18:1-PC via acyl editing in fad2-1 mutant,FAD2 artificial mi RNA and FAD2 co-suppression lines,which was different from the previous conclusion.However,when FAE1 was highly expressed,the relative content of20:1 was increased,and the other saturated and polyunsaturated VLCFA were also increased.Thus the accumulation level of VLCFAs was increased up to 60%.(4)FAE1 could cause the accumulation of the diversified VLCFAs.The types and contents of products in Arabidopsis seeds were determined by the balance between the availibility of substrates and the abundance of FAE1.Results indicated that when FAE1 was highly expressed,apart from its naturally preferred product,20:1,other VLCFA,including22:1,20:0,20:2,20:3,22:0,22:2,22:3 and?24C also accumulated in seeds.However,with the FAE1 high expression,the increased 18:1 availability by knocking-down FAD2singularized the VCLFA products;seeds primarily accumulated 20:1,which increased up to65%,suggesting that further increasing substrate availability could reverse the effect of product diversification due to overabundant FAE1.Re-analysis of FAD2 knockdown lines indicated that increasing 18:1 also led to decreases of 22:1.This suggests that in the presence of overwhelming proportions of the preferred substrate,18:1,the side elongation reactions are suppressed.Collectively,these results demonstrate that a balance between the levels of enzyme and substrate may be the crucial factor for engineering VLCFA products.(5)T.majus can synthesize triacylglycerol with three erucic acyls.To explore the acyltransferase with erucic acyl preference at sn-2 position of triacylglycerol,the function of Tm LPCAT from T.majus was analyzed.The content of erucic acid at sn-2 position of triacylglycerol was increased up to 40 times in the seeds of Arabidopsis(HE)and rapeseed(B.napus)overexpression lines.However,within majority of camelina transgenic lines,no significant increase in the content of erucic acid in sn-2 position of triacylglycerol was observed.Therefore,we concluded that the Tm LPCAT from T.majus has the function of assembling erucic acid in sn-2 position of triacylglycerol.Thus,it can fill in the gaps that erucic acid cannot be effectively bound in sn-2 position of B.napus.(6)MoDGAT2 and Tm GPAT9 improved the accumulation of VLCFAs in A.thaliana.The analysis of fatty acid compositions at the sn-2 position of triacylglycerol showed that the nervonic acid could not be effectively accumulated in this position in seeds of Malania oleifera Chun et S.Lee ex S.Lee,indicating that the nervonic acid mainly accumulated at sn-1 and sn-3 positions.Transcriptome analysis showed that MoDGAT2 was highly expressed.MoDGAT2partially complemented dgat1 mutant function,and significantly increase the accumulation of different VLCFA in high erucic acid lines.Besides,overexpression of Tm GPAT9 in A.thaliana wild-type also increased VLCFA accumulation and oil content.These results suggest that the content of target VLCFA in seeds can be improved by regulating the key genes in the fatty acid synthesis pathway according to their substrate specificity.