| Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao(Astragalus mongholicus)is a perennial herbaceous plant of Leguminosae,which mostly uses dry roots as medicine and has significant pharmacological effects on immunity and anti-tumor.Flavonoids are one of the main active ingredients in Astragalus mongholicus,which have effects in regulating immune system,antioxidation,anti-aging,etc.However,there are few studies on the regulation and molecular mechanism of flavonoids biosynthesis in Astragalus mongholicus,especially on the expression of key genes in biosynthesis and accumulation of flavonoids.The content of total flavonoids in different organs at different periods was determined by UV spectrophotometry.Based on Illumina-Hi Seq platform,the roots of 2-year-old Astragalus mongholicus were sequenced in 5 different growth stages(vegetative stage A,florescence stage B,fruiting stage C,fruit ripening stage D and defoliating stage E).After completing the assembly and annotation,the important metabolic pathway of Astragalus mongholicus was analyzed,and statistical annotation analysis was carried out on the measured data.The main results were as follows:1.The content of total flavonoids in Astragalus mongholicus was determined and analyzed.It was found that the total flavonoids were distributed in different growth periods(vegetative stage,florescence stage,fruiting stage,fruit ripening stage,early deciduous stage,defoliating stage and dormant stage)and organs(root,stem,young leaf,mature leaf,old leaf,flower and fruit).Statistical analysis showed that the content of flavonoids not only varied significantly in different developmental stages of the same organ,but also had significant organ differences.The results showed that the content of flavonoids in the aboveground part of Astragalus was higher than that in the underground part.2.After screening and assembling the original data,158,123 Unigenes with an average length of 644.89bp were finally obtained,of which the longest fragment was 15,637 bp and N50 was 996 bp.The blast program was used to map Unigene sequences to nucleic acid and protein sequence data,among which 8,362 sequences could be annotated by Nr,swiss-prot,egg NOG,GO,KEGG and other public databases.With |log 2(fold change)|≥1 and significance P-value < 0.05 as the standard,differentially expressed genes and transcription factors were screened in the comparison combinations of 4 adjacent periods(A vs B,B vs C,C vs D,D vs E).The GO and KEGG enrichment analysis of the differentially expressed genes showed that the differentially expressed genes in each group were the most annotated into the metabolic process,and contained several biosynthetic entries of secondary metabolites related to flavonoid synthesis.3.Genes related to flavonoid biosynthesis were selected from the differentially expressed genes noted.A total of 86 Unigenes participated in the biosynthesis of isoflavones(12),flavonoids(5)and phenylpropane(69).Further analysis of the expression of these differential genes showed that the expression patterns of the key enzymes PAL,4CL,CCR,CHS,DFR were consistent with the accumulation trend of total flavonoids.Genes related to flavonoid biosynthesis in randomly selected transcriptome data were verified by qRT-PCR.The results showed that qRT-PCR results were consistent with transcriptome data.The above research obtained transcriptome data of Astragalus mongholicus root in different periods,combined with the variation law of flavonoids content,and discussed the relationship between the expression patterns of key enzyme genes PAL,4CL,CCR,CHS,DFR,etc.in flavonoids biosynthesis pathway and the synthesis and accumulation of flavonoids compounds,which laid a theoretical foundation for further using molecular means to regulate the content of secondary metabolites in medicinal plants,thus improving the yidld and quality of medicinal materials. |
PDF Full Text Request