Study On Function Of Peony Tree DGAT3 Gene During Triacylglycerol Accumulation

Tree peony(Paeonia rokii)is a kind of garden flower which is loved by the people with both ornamental value,medicinal value and nutritional value.P.rokii is famous for its rich unsaturated fatty acids,especially α-linolenic acid(ALA)content of more than 40%.However,the lipid accumulation and metabolism mechanism of P.rokii has not been analyzed yet.Acyltransferase DGAT is the rate-limiting enzyme for the transfer of sn-3fatty acids in plant oils.Previous studies have shown that DGAT is involved in the efficient accumulation of Tree peony oils.In this study,one soluble acyltransferase PrDGAT3 was screened from the transcriptome database related to P.rockii previously measured.According to the gene sequences in the database,gene cloning and biological information analysis were performed.Then,PrDGAT3 was used to restore the lipid synthesis of saccharomyces Cerevisiae with lipid synthesis defect(H1246),and the enzyme activity and substrate preference of PrDGAT3 was identified using the principle of yeast lipid toxicity.Finally,the role of PrDGAT3 in the biological synthesis of oil of P.rockii was verified by transient transformation of tobacco,stable transformation of Arabidopsis thaliana and TRV-VIGS,which finally provided theoretical basis for oil synthesis and molecular regulation network of fatty acid metabolism of peony purplifolia.The main research results are as follows:1)The diacylglyceryl transferase gene PrDGAT3 was cloned from the Tree peony of purple patch.The complete coding region of PrDGAT3 was 1173 bp,encoding a protein with 390 amino acid residues.Subcellular localization experiments showed that the protein was localized to chloroplasts.The expression level of PrDGAT3 in different tissues of Tree peony was detected by q RT-PCR assay.The results showed that the expression level of PrDGAT3 was the highest in stems and the second in leaves.2)PrDGAT3 was expressed in a tetraplex yeast mutant(H1246)with defects in TAG biosynthesis.Lipid recovery test showed that in the yeast expression system,the experimental results showed that DGAT3 of Tree peony had enzyme activity and could transfer oleic acid(C18:1),linoleic acid(C18:2)and α-linolenic acid(C18:3)fatty acids.Moreover,it has more substrate bias for C18:3.3)The PrDGAT3 gene of peony was expressed instantaneously in the leaves of the tobacco,and the oil synthesis of the leaves was observed by dyeing the oil with Nile red.Compared with the control leaves,the number of oil drops in the leaves transferred with PrDGAT3 gene was significantly increased.By gas chromatography analysis,the total fatty acid content in transgenic tobacco leaves was significantly increased compared with the control leaves,especially the proportion of C18:2 and C18:3 in transgenic tobacco leaves.4)Stable expression of PrDGAT3 gene of P.rockii in wild-type Arabidopsis thaliana(Col-0),transformation of Agrobacterium strain GV3101,inflorescence impregnation of PrDGAT3 into wild-type Arabidopsis Col-0 plants,through continuous seed collection,antibiotic screening and plant growth process,The T3 generation pure transgenic plants were identified by PCR.The fatty acid content in T3 rosette leaves was determined by gas chromatograph.Compared with blank control leaves,the proportions of C18:2 and C18:3in transgenic leaves were increased,especially the proportions of C18:3 were significantly increased.5)Through reverse genetic verification,PrDGAT3 gene based on tobacco cracking virus(TRV)VIGS gene was silencing on the body of P.rockii spot,and TRV2-PrDGAT3 was transiently transformed into leaves of peony seedlings by vacuum osmosis.Under ultraviolet lamp observation,green fluorescence appeared in the leaves of momentary transformation of peony seedlings,and bands were observed by semi-quantitative PCR.Real-time fluorescence quantitative q RT-PCR analysis showed that the expression level of PrDGAT3 was significantly decreased compared with blank control leaves.By gas chromatography analysis of fatty acid content in leaves of peony seedlings,the proportion of α-linolenic acid(C18:3)in leaves of peony seedlings with silence of PrDGAT3 was significantly decreased.The proportion of linoleic acid(C18:2)was significantly decreased.