Cloning And Functional Identification Of Key Genes Involved In Oil Body-associated Proteins And The Synthesis Of Sciadonic Acid In Torreya Grandis

Torreya grandis,is an evengreen gymnosperm belonging to the family Taxaceae.T.grandis’Merrillii’ is mainly reproduced by asexual mode of reproduction.In China,the kernels(dry fruits)of T.grandis have high nutritional values,which are main source of rich unsaturated fatty acids.T.grandis kernels contain a special unsaturated fatty acid,Sciadonic acid(20:3Δ5,11,14,SA),which are famous for its high economic and medicinal values.However,the molecular basis of the synthesis and regulation of SA and oil in T.grandis still remains unclear.In our research,10 landraces and 5 developmental stages of T.grandis kernels were selected.The key genes involved in the metabolic pathway of oil and SA were identified by RNA-Seq and bioinformatic analysis.Furthermore,a series of molecular,physiological and biochemical experiments were applied in our research.Besides,preliminary functional analysis of some key genes has been carried out.The main results are mentioned as follows1.The metabolic pathway of oil and SA has been identified by RNA-Seq of T.grandis kernels in 10 landraces and 5 developmental stages.According to the determination of oil content and fatty acids composition,it is found that the oil content varied apparently in different landraces,and increased continuously with the expansion of the fruit.T.grandis contains six main unsaturated fatty acids,among which oleic acid and linoleic acid are the most abundant,while the following is SA,up to 53 mg·g-12.Via evolutionary tree analysis and amino acid sequence comparison,three genes TgOLEO1,TgCLO1 and TgSLO1,which encoding oil body-associated proteins Oleosin,Caleosin and Steroleosin,were screened and amplified,with the product sizes of 414bp,699bp and 1017bp respectively.Subcellular localization analysis in epidemic cells of tobacco leaves showed that TgOLEO1-GFP,TgCLO1-GFP and TgSLO1-GFP are located in ER and oil body,and overexpression of them could increase the amount of oil bodies,while overexpression of TgSLO1-GFP could also change the size of oil body.3.Via evolutionary tree analysis and amino acid sequence comparison,two genes,TgELO1 and TgDES1,which encoding Δ9 fatty acid elongase and Δ5 fatty acid desaturase related to the synthesis of SA were screened and amplified,with the product sizes of 846bp and 1242bp respectively.Subcellular localization analysis of TgELO1-GFP and TgDESl-GFP in epidemic cells of tobacco leaves showed that both of them are located in ER.The results of fatty acids determination showed that T.grandis could use linoleic acid and eicosaenoic acid as substrates to synthesize SA under the catalysis of Δ9 fatty acid elongase and A5 fatty acid desaturase.