| Helianthus annuus L.is one of the four major oil crops in the world.Improving oil content and modifying fatty acid composition of sunflower seeds are important goals of sunflower quality breeding.The biosynthesis of plant lipid involves multi-gradation and multilevel synergistic regulation,but the molecular regulation mechanism of plant lipid metabolism is still not well studied.In this study,we firstly analyzed the variation trend of weight,moisture content,oil content and fatty acid composition of three sunflower maintainer seeds at different development stages,which provided theoretical basis for molecular mechanism study of seed development and quality formation in sunflower.Whole transcriptomic sequencing was carried out using sunflower seeds with high oleic acid content at three representative developmental stages,and the genes and the non-coding RNAs involved in lipid metabolism were explored,which provided reference for elucidating the molecular regulation mechanism of lipid metabolism during sunflower seed development.Gene function analysis and upstream transcription factor prediction of HaFAD2,a key candidate gene controlling fatty acid content in sunflower,provided gene resources for improving sunflower quality by molecular means,and laid a foundation for future studies on the transcriptional regulation mechanism of HaFAD2.The main results are as follows:1.The analysis results of weight and moisture content of sunflower seeds during development showed that the fresh weight of 100 seed kernels,100 seed husks and 100 seeds of three sunflower maintainer lines all accorded with the trend of first increasing and then decreasing,but the time points of the change trend of different materials were different.The fresh seed kernel rate,the dry weight of 100 seed kernels,the dry weight of 100 seeds and the dry seed kernel rate increased gradually in general;The dry weight of 100 seed husks increased in the early stage of seed development,and then it changed slightly.The moisture content of seeds increased at 7-12 d after flowering,decreased gradually at 12-32 d after flowering,and decreased more significantly at the later stage of development.2.Analysis of oil content and fatty acid composition during seed development of sunflower showed that the variation trend of oil content of three materials is basically consistent,with the maximum accumulation rate occurring at 17-32 d after flowering,and the oil content tended to be stable at 32-37 d after flowering.The relative contents of stearic acid(C18:0)and linolenic acid(C18:3)were very low during the whole seed development.The content of palmitic acid(C16:0)was higher in the early stage of seed development,but decreased with seed development.The accumulation trend of oleic acid(C18:1)and linoleic acid(C18:2)was basically opposite.The difference of oleic acid accumulation between high and low oleic acid sunflower was as follows:oleic acid in high oleic acid sunflower mainly accumulated rapidly at 7-12 d after flowering,while oleic acid in low oleic acid sunflower accumulated gradually at 12-22 d after flowering.The contents of 37 kinds of fatty acids were detected in sunflower seeds with high oleic acid content at 7,22 and 37 d after flowering,and the results showed that 13 kinds of fatty acids were detected,of which the types of fatty acids at 22 d were the most and 37 d were the least.3.MRNAs,lncRNAs and circRNA were sequenced on sunflower seeds with high oleic acid content at 7,22 and 37 d after flowering,a total of 237,767 transcripts were detected.52293 mRNA,120625 lncRNA and 2723 circRNA were in the L7d vs L22d comparison group,57398 mRNA,123485 IncRNA and 2910 circRNA were in the L7d vs L37d comparison group,while 15417 mRNA,9554 lncRNA and 1538 circRNA were in the L22d vs L37d comparison group as for the standard of Fold Change≥2 and Pvalue≤0.001.KEGG metabolic pathway enrichment analysis revealed that some differentially expressed genes,lncRNA target genes and circRNA-derived genes were classified into lipid metabolism related pathways in all three groups.In particular,circRNA-derived genes were significantly enriched in fatty acid metabolism related pathways,suggesting that circRNA might play an important role in regulating gene expression in fatty acid metabolism pathways.Analysis of expression heat map revealed that most of DEGs in fatty acid biosynthesis pathway were down-regulated in three comparison groups,which suggested that most of these genes were down-regulated with seed development.QRT-PCR of 8 DEGs selected from lipid synthesis pathway showed consistent expression trends with transcriptomic sequencing detection,which verified the reliability of whole transcriptomic sequencing results.4.MiRNA were sequenced on sunflower seeds with high oleic acid content at 7,22 and 37 d after flowering,a total of 268 miRNAs were identified,including 98 new miRNAs.95,127 and 77 miRNAs were differentially expressed in L7d vs L22d,L7d vs L37d and L22d vs L37d groups respectively as for the standard of Fold Change≥2 and Pvalue≤0.001.There were 22 differentially expressed miRNAs in lipid metabolism related pathways among the three groups.Analysis of expression heat map showed that most of miRNAs in the 22d vs 37d comparison group were down-regulated.Co-expression analysis of differentially expressed miRNAs and target genes showed that 56(L7d vs L22d),79(L7d vs L37d)and 31(L22d vs L37d)differentially expressed miRNAs were negatively correlated with the target genes in the three comparison groups,in which,there were 6 pairs of miRNAs and the corresponding target genes related to lipid metabolism,indicating that the differential expression of these genes may be the result of miRNA regulation.By analyzing the regulatory network of ceRNA related to lipid metabolism,we found some key enzymes that may be regulated by ceRNA network,including glycerol-3-phosphate acyltransferase,linoleic acid 9s lipoxygenase and so on.5.16 genes encoding key enzymes in fatty acid and triglyceride biosynthesis pathways were selected,and the expression of these genes in sunflower seeds at different stages of development,leaves,stems,tubular flowers,ray flowers and roots were detected by qRT-PCR.It was found that only FAD2 was a specific high expression gene in seeds.The results of correlation analysis showed that there was a significant correlation between C16:0 and the expression of KASⅢ,C18:1 and the expression of LPCAT,MCAT,FAD2,KASIII,C18:2 and the expression of FAD2,KASⅢ,C18:3 and the expression of KASⅢ.Oil content was significantly correlated with the expression of EAR,DGAT3,LPCAT,PAH1,FAD2,MCAT and LACS4,and there was no significant correlation between C18:0 and the expression of these 16 genes.6.The expression vector 2301-HaFAD2-GFP was constructed and transiently transformed into the lower epidermis of tobacco leaves.HaFAD2 coding protein was found to be subcellular localized in the cytoplasm.The overexpression vector 2301-HaFAD2 was constructed and infected with Arabidopsis thaliana by floral dip method.The transgenic positive seedlings were identified by PCR and qRT-PCR after root culturing and screening by Kana.Four transgenic lines with differential expression were selected for gene function analysis.Compared with the wild type,the oil contents of the seeds of the transgenic HaFAD2 strains were significantly increased,the fatty acid composition changed,and the total unsaturated fatty acid content was increased.The content of most fatty acids in the downstream of linoleic acid synthesis were significantly increased,while the content of most fatty acids in the upstream of linoleic acid synthesis was significantly decreased.HaFAD2 promoter with 867 bp nucleotides sequence was cloned.According to the prediction of potential cis acting elements in the promoter region,the cluster heat map and correlation analysis of the expression between HaFAD2 and transcription factors,it was speculated that bHLH96 was likely to be a transcription factor regulating HaFAD2 upstream. |
PDF Full Text Request