| Cottonseed oil is the main byproduct of cotton.Cottonseed oil contains high level of unsaturated fatty acids(UFA),which is suitable as edible oil.Phosphoenolpyruvate carboxylase(PEPC)mainly catalyzes phosphoenolpyruvate to yield oxaloacetate(OAA),which was involved in the tricarboxylic acid(TCA)cycle.However,PEPC is also involved in regulating the distribution of carbon flow.In present study,cottonseed developing embryo at 10 DPA,20 DPA and 30 DPA were used for transcriptome sequencing,and gene expression network of cottonseed oil accumulation was established.Evolution and expression analysis of PEPC gene family were carried out in the four genome sequenced cotton species,and the gene duplication events and functional diversity of PEPC gene during evolution were proposed.In addition,the transcriptome datasets of cottonseed embryos in GhPEPC2 interfering transgenic lines and the control were used for transcriptome sequencing,and the molecular basis of increased cottonseed oil content by RNA interfering of GhPEPC2 was analyzed.At the same time,some candidate genes involved in TAG biosynthesis were cloned and transformed into tobacco,and genes function involved in oil accumulation was verified initially.Transgenic materials with high oil content were screened and used for stresses analyses,in which the resistance of cotton seedlings in high oil content transgenic materials was significantly enhanced.In short,gene expression network of oil accumulation reveals the reason of high linoleic acid content in cottonseed oil;the evolution and expression analysis of PEPC genes provides the basis for revealing its influence on carbon flow.The main results are as follows:1.In developing cotton embryos,totally 8,629,7,891 and 12,555 differentially expressed genes(DEGs)were detected in the comparison sets of '20 DPA vs 10 DPA','30 DPA vs 20 DPA' and '30 DPA vs 10 DPA',respectively.Gene network described the dynamic expression profiles of oil accumulation in fatty acids(FAs)synthesis,FAs desaturation and triacylglycerol(TAG)biosynthesis.WRI1 and NF-YB6 were suggested elite transcription factors in regulating lipid metabolism in cottonseed.Meanwhile,GhPDAT was highly expressed and might play more important role than GhDGAT in transforming diacylglycerol to TAG.Expression patterns of twelve FA biosynthesis-related genes were randomly selected and validated by quantitative real-time PCR(qRT-PCR)method.To reveal the reason of the high content of linoleic acid(C18:2)in cottonseed oil,we carried out comparative analysis of genes expression levels in Upland cotton,rapeseed(Brassica napus)and oleaster(Olea europaea).Compared with in rapeseed and oleaster,GhFAD2 genes were up-regulated,and GhFAD3 genes were down-regulated in cottonseed,which accounted for the relative high amount of C18:2 but low content of linolenic acid(C18:3)in cottonseed oil.2.We identified 6,6,11 and 10 PEPC genes in Gossypium raimondii(D5),G.arboreum(A2),G.hirsutum(AD1)and G.barbadense(AD2),respectively.The 33 PEPC genes were divided to 6 subgroups in cotton,each subgroup showed conserved gene structure and motifs.Gene chromosomal location showed that orthologs PEPC genes in tetraploid cotton were conserved in At and Dt subgenomes(t represents tetraploid),and orthologs were derived from A and D genomes.Whole genome duplication(WGD)and segmental duplication events yield the expansion of PEPC genes.The duplicated PEPC genes showed different expression patterns in cotton tissues,indicating that they might experience functional divergence in cotton evolution.Gene expression patterns under multiple stresses displayed that GhPEPC genes were induced by various treatments,and genes expression of GhPEPC1 and GhPEPC2/GhPEPC2D were dramatically elevated under multiple stress treatments.3.Gene expression profiles in developing cottonseeds of GhPEPC2 interfering transgenic lines and the control were investigated using RNA-Seq approach.In transgenic lines,a number of lipid metabolism related GO terms and KEGG pathways were significantly enriched in up-regulated DEGs,whereas amino acid metabolism related GO terms and KEGG pathways were significant enrichment in down-regulated DEGs.Meanwhile,many genes related to lipid synthesis were up-regulated,whereas TCA cycle connected genes were down-regulated expression in transgenic lines.4.Gene network on cottonseed oil accumulation showed that the key enzyme encoding genes in TAG synthesis were up-regulated with cottonseed development,transcriptome analysis on high and low oil content cotton materials displayed that these genes were higher expressed in high oil content cottonseed.The candidate genes were cloned and used for overexpression vectors construction.Then,the recombinant vectors were transformed to tobacco by leaf disc method.Overexpression of these genes increased the oil content of tobacco seeds in different levels,oil content of tobacco seed in overexpression of GPDH transgenic lines increased 13.54%compared with the control,suggesting the important function of these genes in oil accumulation.5.The previous transgenic cotton materials with high oil content were used for further screening.Finally,oil content in cottonseed of GhPEPC2 interfering transgenic lines increased 28.51%,oil content in over-expression of GhENR,GhKAR and GhHAD transgenic lines increased 14.58%,20.36%and 10.32%compared with the control,respectively.Germination test and seedling root scanning experiments on the transgenic lines and the control under stress conditions were performed.The results showed that the resistance of transgenic plants were enhanced,which the seedlings salinity and cold tolerance in GhPEPC2 interfering transgenic lines were significantly enhanced in comparison to the control,indicating that the increasing of seed oil content could enhance cotton resistance to abiotic stresses. |
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