Cloning And Expression Analysis Of Key Enzyme Genes Involved In Storage Lipid Metabolism Of Chinese Dwarf Cherry[Cerasus Humilis(Bge.)Sok.]

Chinese dwarf cherry[Cerasus humilis(Bge.)Sok.],a small deciduous shrub is a member of the family Rosaceae and originated in the North of China.It is characterised by its high level of resistance to drought and cold,the ability to tolerate barren soils and the easiness to cultivate.It is a very promising drupe fruit tree for arid area.The kernels of Chinese dwarf cherry,which are rich in unsaturated fatty acids,can be used for high quality food oil or used as raw material for the industry.The fatty acid composition of Chinese dwarf cherry is quite similar to olive oil.In this study,the key enzyme genes involved in storage lipid metabolism were studied based on the RNA sequencing of the kernels in three different developmental stages.Expression of the genes was also conducted to analyze the relation between gene expression and lipid accumulation.The main findings are as follows:1.Seeds of three different developmental stages from cultivar ’Nongda No.4’ were used as the material for the RNA sequencing through the Illumina Hi-Seq2000 platform.The three stages are early stage(6-1 A),middle stage(6-2A)and late stage(6-3A)respectively.Fruit transcriptome database of Chinese dwarf cherry were used as the reference.In total,29348,24559 and 32890 unigene sequences were annotated respectively from the sequencing output of ’6-1A’,’6-2A’ and ’6-3A’.The numbers of unique unigene sequence in the outputs of’6-1 A’,’6-2A’ and ’6-3 A’ were 803,228 and 3673 respectively.Most redundant sequences were 200-3000 nucleotides in length and the number of sequences that longer than 3000nt from three stages were 1146,1078 and 1178.The total amount of unigenes annotated in the common database(NR,NT,Swiss-Prot,KEGG,COG and GO)were 31726.18230 sequences were annotated by KEGG database and 127 metabolic pathways were involved and 1729 unigenes were annotated to 14 lipid metabolism associated pathways.2.The sequencing results of three different stages were compared with each other.Compared to 6-1A,4078 unigenes were differentially expressed in 6-2A(2439 were up-regulated,2269 were down-regulated);7883 unigenes were differently expressed 6-3A versus 6-1A(5446 were up-regulated,2437 were down-regulated);5992 unigenes were differently expressed 6-3A versus 6-1A(3842 were up-regulated,2151 were down-regulated).In terms of GO Biological Process,differentially expressed genes were mainly enriched in metabolic process and cellular process;in terms of Cellular Component,differentially expressed genes were mainly enriched in Cell,Cell Part and Organelle;in terms of Molecular Function,differentially expressed genes were mainly enriched in Binding and Catalytic Activity.3.KEGG pathway annotation showed that differentially expressed genes were mostly distributed in the Biosynthesis of secondary metabolites and the Metabolic pathways.There were 25 unigenes related to the fatty acid biosynthesis.These 25 unigenes represent 13 fatty acid biosynthesis related genes after blasting in KEGG database.From 6-1A to 6-2A,12 genes were up-regulated which showed that large quantity of fatty acids were synthesized during this developmental stage.SAD gene,which catalyses the accumulation of oleic acid,had the highest RPKM value(>3000)among all genes that related to fatty acid accccumulation metabolic pathways.FAD2 gene,which catalyses the tansformation of oleic acid to linoleic acid,also had high expression level(RPKM value>2000),while FAD3,which catalyses the tansformation of linoleic acid to linolenic acid,had very low RPKM value(<100).The expression of all three genes contributed to the accumulation of oleic acid and linoleic acid in the seed of Chinese dwarf cherry.There were 23 unigenes related to the Glycerolipid metabolism which represent 6 storage lipid biosynthesis related genes.From 6-1A to 6-3A,5 genes were up-regulated which showed that large quantity of newly synthesized fatty acids were assembled into glycerol backbone to form TAG and then TAG were stored in the seed kernels.The expression levels of the genes that associated with acyl-editing pathway were very low which showed that the dominated pathway of TAG biosynthesis is de novo assembly of TAG,which is the Kennedy Pathway.4.Based on the pathway analysis of differentially expressed genes,10 key genes that participate in fatty acid biosynthesis and TAG assembly were selected.These genes were a-CT,BCCP1,BCCP2,fatA,SAD,FAD2,FAD3,GPAT,DGAT and PDAT.Complete coding sequences were obtained by assembling all unigenes that from the same gene.Primers were designed for each gene and Reverse transcription PCR were carried out.The PCR amplifications were put into T-easy vector and positive clones were sent for sequencing.The sequencing results showed that the sequences of positive clones were matched with the predicted sequence and proved that the genes cloned in this study were right.The structures and functions of the proteins encoded by these 10 genes were predicted and analyzed by using online tools.Protein sequences were blasted with published sequences from other plant species and the results showed that these proteins in Chinese dwarf cherry have high similarity with other oil crop plants.5.The oil contents and fatty acid compositions from 31 genotypes of Chinese dwarf cherry were evaluated.The results showed Chinese dwarf cherry has high potential to be a new oil crop because of its high seed oil contents and favorable fatty acid composition.Five fatty acids in the kernel oil were detected,which were Palmitic acid(C16:0),Palmitoleic acid(C16:1),Stearic acid(C18:0),Oleic acid(C18:1)and Linoleic acid(C18:2).Oleic acid and Linoleic acid were the main fatty acids and accounted for 67.0%and 28.6%of the total fatty acid.Oil content correlated negatively with palmitic and stearic acids,and positively with oleic acids.The correlations we found in this study may shed light on the future selection and breeding of Chinese dwarf cherry.6.Cultivar ’Nongda No.4’ of Chinese dwarf cherry were used for kernel oil and fatty acid accumulation study and real-time quantitative PCR analysis of 10 storage lipid biosynthesis related genes.The relation between gene expression and fatty acid accumulation was analyzed.The results showed that the quantitative PCR results were consistent with the sequencing results and both of them were corresponding to the accumulation of fatty acids and the storage lipid.Fatty acid biosynthesis and TAG assembly pathways were defined in this study.The expression patterns of storage lipid biosynthesis related genes were studied together with the relations between gene expression and its metabolic products.This work lays the foundation for future developmental regulation study of Chinese dwarf cherry seeds and provides some basic reference theories for the further study of lipid metabolism and breeding(especially in molecular breeding).