| The main characteristic component of the fruit of Citrus aurantium L.is the phenethylamine alkaloid synephrine,which is one of the important indicators to measure the quality of C.aurantium.Since the content of the phenethylamine alkaloid synephrine in C.aurantium fruit is very low,it is difficult to extract and prepare a large amount of the alkaloid synephrine by traditional separation methods.Therefore,it is urgent to explore a new method that can obtain the alkaloid synephrine in large quantities.In this study,high-throughput transcriptome sequencing technology was used to mine the key genes that regulate the synthesis of the phenethylamine alkaloid synephrine in C.aurantium.And metabolic engineering to efficiently produce medicinal components,C.aurantium germplasm improvement and variety cultivation provide a theoretical basis.The main research contents and results are as follows:In this study,the HPCE method was optimized and used for the determination of synephrine content in Yuanjiang C.aurantium fruit samples to provide reference for quality evaluation and control,and determination of the optimal harvest time.The method is simple,efficient and accurate,which is suitable for the determination of synephrine content in Yuanjiang C.aurantium fruit.Drawn from the determination results,the harvest time had a significant effect on the content of synephrine in C.aurantium fruit（P < 0.05）.The optimal harvest time should be from May to mid June for the immature fruit of Yuanjiang C.aurantium,and from mid July to mid August for the mature fruit.The content of synephrine in 21 C.aurantium germplasms（harvested on May 18th）ranged from 7.64 to 9.96 mg/g,and the content of synephrine in code 9,21,50,65 and 77 C.aurantium germplasms was higher,which could provide excellent germplasm resources for breeding C.aurantium varieties with high alkaloid characteristics.GO and KEGG analysis of differently expressed genes: Metabolic processare enriched with the most differential genes.In the cellular component ontology,the organelle and extracellular membranes have the most enriched.In the molecular function ontology,nucleic acid-binding transcription factor activity,catalytic activity and transcription factor activity are significantly different from each other.Protein binding enriched the most differential genes,and transcription factor family genes may play an important regulatory role in the development of C.aurantium fruit and the synthesis of secondary metabolites.KEGG enrichment analysis found that the most differently expressed genes were enriched in metabolic pathways,and the metabolic pathways related to the synthesis of C.aurantium phenethylamine alkaloids were glycolysis/gluconeogenesis pathway（ko00010）,pentose phosphate pathway（ko00030）,Phenylalanine,acid tyrosine and tryptophan biosynthesis pathway（ko00400）and tyrosine metabolism pathway（ko00350）.Candidate gene screening: According to literature reports and KEGG annotation analysis of DEGs in the three comparison groups,the key enzyme genes related to the synthesis of C.aurantium phenethylamine alkaloids are: ENO,tkt,Aro F/G/H,aro A and DDC,They are involved in the regulation of glycolysis/gluconeogenesis,pentose phosphate pathway,phenylalanine tyrosine and tryptophan biosynthesis,and tyrosine metabolism pathways,respectively.Transcription factor prediction:The i TAK software was used to predict the transcriptome DEGs transcription factors of the C.aurantium C17-1/C17-7comparison group,involving a total of 20 TFs,of which the MYB transcription factor family had the largest number of genes,and a total of 46 differences were predicted,There are 21 up-regulated genes and 25 down-regulated genes.Gene expression trend analysis: Through gene expression trend analysis,new key enzyme genes aro B,aro C and CM were identified,which are involved in regulating the synthesis of aromatic amino acids in the biosynthetic pathways of phenylalanine,tyrosine and tryptophan. |
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