Identification Of The Key Gene FAE1 With High Erucic Acid Synthesis Activity And Its Genetic Transformation Into Rapeseed(Brassica Napus L.)

VLCFAs(Very long chain fatty acids)are fatty acids with more than 18 carbon atoms in the main chain.They exist in organisms in four forms: triacylglycerol(TAG),wax precursors,glycerophospholipids and sphingolipids,and are involved in a wide range of life processes.They are important structural components of living organisms and are also important functional substances in the life activities of organisms,such as resistance to adversity,defence against disease,signal transduction and energy storage.However,due to its genetic composition,oilseed rape lacks a functional LPAT2 that introduces erucic acid(C22:1)at the sn-2 position in TAG,so the limit of erucic acid content in its seeds is 66.7%.To break through this theoretical value,genetic engineering is the obvious choice.In this study,FAE1,a key gene for erucic acid synthesis,was first cloned from five plants with different seed erucic acid contents and then compared in a yeast expression system for its erucic acid synthesis capacity.Finally,a combination of FAE1 and Ld LPAT2 with high erucic acid synthesis capacity was selected to construct a seed-specific expression vector,and Agrobacterium-mediated transformation was used to genetically transform oilseed rape in order to obtain(ultra)high erucic acid rapeseed germplasm material.The main findings of this study are as follows:1.The homologous cloning method was used to obtain 12 FAE1 genes,which normally encode proteins,from five plants,including high erucic acid rapeseed,high oleic acid rapeseed,Crambe abyssinica,Tropaeolum majus and Limnanthes douglasii.Sequence analysis showed that the copy numbers of FAE1 genes were inconsistent in different plant genomes,and that there were SNP sites between functional genes and differences in the encoded amino acid sequences.The sequence identity of the 12 FAE1 genes ranged from 52.1% to 99.9% for c DNA and from 49.9% to 99.8% for the deduced amino acid sequences.The FAE1 gene is clearly species-specific.2.Recombinant yeast expression vectors for each of the above 12 FAE1 genes were constructed using plasmid p YES2/NT A as a backbone and analysed for induction of expression and comparative erucic acid content.GC-MS analysis results showed that eight FAE1 genes derived from Mianyou328,Crambe abyssinica and Limnanthes douglasii had the ability to synthesize VLCFAs,of which Ca FAE1-3 having the strongest ability(4.82%),followed by Gj FAE1-1(4.53%),while Ld FAE1 had the weakest ability(0.29%).The other four FAE1 genes derived from Yangguang80 and Tropaeolum majus were all non-functional because Gy FAE1-2,Tm FAE1-1 and Tm FAE1-2 had mutations in the conserved cysteine or(and)histidine sites,while Gy FAE1-1 had an R395 K mutation resulting in loss of enzymatic activity.3.A seed-specific expression vector was constructed with Ca FAE1-3 and Ld LPAT2 using p CAMBIA1300 as the backbone.Two positive transgenic seedlings were obtained by Agrobacterium-mediated transformation using Mianyou328 as the recipient.Taken together,the results of this study will improve the understanding of the relationship between structure and function of the FAE1 sequence,and will highlight the identification of the key amino acid sites that determine or influence substrate specificity and elongase activity,and will also be beneficial for genetic engineering breeding of(ultra)high VLCFAs content.