| Camellia oleifera Abel.is a very important woody tree specie for producing edible oil which contains rich unsaturated fatty acids and the best composition proportion of fatty acids.It is a kind of closely watched high-quality edible oil at present.In this study,the seed kernels of C.oleifera at three periods were selected as experimental materials on Oct.24th (P1),Nov.7th (P2) and Nov.21nd (P3) in 2015.The transcriptome of seed kernels at the three periods was analyzed by using the high-throughput sequencing platform Illumina Hiseq X ten.Water content,oil content and composition of fatty acid in seed kernels were measured.Together with the determination of enzyme activities and the mRNA expression levels of genes related to lipid metabolism using RT-qPCR,the molecular regulation mechanism of lipid metabolism was investigated systematically during maturation of C.oleifera seeds.(1) The results of water and oil content at the three periods showed that,the water contents were decreased and the oil contents were increased with seed maturation,and the oil content reached the 47.90% in P3 stage,suggesting that the lipid biosynthesis was always active to increase the oil content of seeds during its maturation of C.oleifera.(2) After analysis of the fatty acid composition of C.oleifera oil by using GC-MS,the results showed that nine fatty acids were isolated stably from seed kernels at the three periods,among which the main component of fatty acid was oleic acid (about 80% of fatty acid content).At each period,the content of oleic acid was much higher than the other fatty acids and the content of unsaturated fatty acid also significantly higher than saturated fatty acid.In the process of P1 to P2,the contents of oleic acid and unsaturated fatty acid were increased significantly,but the contents of linoleic acid and saturated fatty acid were decreased significantly.In the process of P2 to P3,the content of oleic acid decreased significantly,but the linoleic acid increased significantly.(3) The results of transcriptome in C.oleifera seed kernels showed that a total of 24.00 Gb clean data,123,157 transcripts and 75,940 unigenes were obtained.Compared to P1,407 differentially expressed genes were identified,among which 204 genes were up-regulated and 203 genes down-regulated in P2.Compeared to P2,423 differentially expressed genes were identified,among which 286 genes were up-regulated and 137 genes down-regulated in P3.Compared to P1,893 differentially expressed genes were identified,among which 486 genes were up-regulated and 407 genes down-regulated in P3.The findings indicated that the seeds of C.oleifera initiated more metabolic pathways to regulate the growth and development from P1 to P3.By KEGG pathway analysis,fourteen pathways related to lipid metabolism were found,containing glyoxylate metabolism,glycerol phospholipid metabolism,unsaturated fatty acid biosynthesis,fatty acid metabolism,fatty acid synthesis,fatty acid degradation pathway,glycerol ester metabolic pathway,?-linolenic acid metabolic pathway,linoleic acid metabolic pathway,fatty acid prolongation pathway,and so on.Nineteen key genes related to lipid metabolism were found,among which ten genes related to oil synthesis were up-regulation and three genes related to glycolysis showed no changes during seed maturation.(4) The analysis of RT-qPCR showed that the mRNA expression levels of key genes related to fatty acid and lipid synthesis pathways,including ACCase ?-CT,KAS?,LACS2,GPAT4 and FAD2,all were up-regulated with seed maturation,which was consistent with transcriptomic data.The activities of some key enzymes like ACCase and FAS were increased during seed maturation.These results indicated that the up-regulated expression of the key genes involved in fatty acid and lipid synthesis pathways contributed to the increase of the oil content.There was no difference in the expression of FAD2 between P1 and P2,and the expression level of FAD2 was increased in the process of P2 to P3,suggesting that the up-regulation of FAD2 expression promoted the transformation of oleic acid to linoleic acid.Taken together,during seed maturation of C.oleifera,the seeds should always synthesize fatty acids to accumulate lipid,and regulate the quality of tea oil through the conversion of unsaturated fatty acids such as oleic acid and linoleic acid.In a word,this study revealed the molecular mechanism of lipid metabolism of "Min43" C.oleifera during seed maturation.These results will provide a theoretical basis for the breeding of C.oleifera with high oil content and high-quality oil by molecular technique in the future. |
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