| Shikimic acid has anti-influenza,antibacterial and anti-inflammatory effects,and is one of the raw materials of the anti-influenza virus drug "Tamiflu",which comes from the shikimic acid pathway.With the increasing demand for influenza drugs,there is an urgent need to develop new shikimic acid source plants,and ginkgo biloba has become an important potential source tree species of shikimic acid with high shikimic acid content,large leaf yield and wide distribution.In addition,ginkgo biloba is recognized as a plant with a high content of flavonoids,which come downstream from the shikimic acid pathway.In the past,there was a lack of germplasm evaluation and screening based on shikimic acid content,and there was a lack of correlation studies on the ginkgo shikimic acid-flavonoid pathway.This study used 33 primary selection potential leaf ginkgo single plants from the germplasm resource bank of Nanjing Forestry University as materials to analyze the content of the main metabolites of the shikimic acidflavonoid pathway(shikimic acid,phenylalanine,tryptophan,tyrosine,total flavonoids),the interplant variability and correlation of leaf phenotypic traits(single leaf dry weight,single leaf area,specific leaf dry weight),and targeted screening of specific single plants through cluster analysis.Based on the metabolome and transcriptome analysis of shikimic acid and shikimic acid single plants,the interaction relationship between shikimic acid metabolism and downstream metabolism was explored.The main findings are as follows:(1)The phenotypic traits of ginkgo biloba(single leaf area,single leaf dry weight,specific leaf dry weight)and the content of major metabolites(shikimic acid,phenylalanine,total flavonoids,tryptophan,tyrosine)in the shikimic acid pathway were very different among the 33 single plants(p<0.01),and the coefficients of variation of indexes exceeded 10%,showing extensive genetic variation,which improved the germplasm base for screening single plants with high leaf yield or high main metabolite content in shikimic acid pathway.(2)There was a significant positive correlation between tryptophan and tyrosine,tyrosine and total flavonoids,single leaf area and single leaf dry weight(p<0.01),shikimic acid content and specific leaf weight(p<0.01),tyrosine content and specific leaf weight(p<0.05),shikimic acid content and total flavonoids,tryptophan,There was a significant negative correlation between tyrosine content(p<0.05)and a significant positive correlation between single leaf dry weight and specific leaf weight(p<0.01),which could predict the content of major metabolites through simple and easily measured leaf phenotypic traits,which provided a reference for early breeding of single plants with high main metabolite content.(3)Based on the clustering analysis of leaf phenotypic traits and the content of major metabolites in the shikimic acid pathway,when the European distance was 11,the 33 single plants were divided into 4 groups,and the first group only had a single plant 31#,and the total flavonoids,tryptophan,tyrosine content and specific leaf weight were the highest values in the four groups,which were higher than the average values of 25.034%,22.377%,36.531% and 15.369% of the four groups,respectively.The second group included 15#,9#,24#,12#,and the mean content of phenylalanine was the highest,which was higher than the average of 18.954% in the four groups;The third group included 5#,14#,26#,1#,4#,and the average shikimic acid content and single leaf area were the highest levels,which were higher than the average values of 25.231% and 32.249% in the four groups.The fourth group included all but groups 1,2 and 3,and there were no outstanding indicators.(4)Metabolome analysis based on the highest shikimic acid content plant 1# and the lowest single shikimic acid content plant 24# showed that it was shown,a total of 290 differential metabolites were detected.162 metabolites were down regulated and 128 metabolites were up regulated in 1 # vs 24 #,among which HMDB annotated that the category containing the most metabolites was flavonoids and their glycosides(19),followed by terpenoids(16),amino acids,peptides,and analogues(16).Among the top ten differential metabolites with positive and negative values in LOG2 FC,8 come from the shikimic acid pathway,and most of the substances downregulated by 1 # compared to 24 # are flavonoids.Combining HMDB annotations and the top ten metabolites with positive and negative differences in LOG2 FC indicates a strong correlation between shikimic acid content differences and downstream substance metabolism.In the shikimic acid phenylpropane flavonoid pathway,12 differential metabolites were detected in the metabolome: tryptophan,tyrosine,Siberian larch flavone,eugenol,chloranthin,melatonin,kynurenic acid,indole acetic acid,indole acetamide,indole,catechin,luteolin.Combined with correlation analysis of substances between individual plants,it is speculated that the accumulation of shikimic acid is due to the lower generation of downstream substances(downregulation of aromatic amino acid content).Combined with the correlation analysis of inter-plant substances,it is speculated that the low accumulation of shikimic acid in 24# is due to the synthesis and accumulation of flavonoids such as tryptophan,tyrosine and luteolin.(5)Transcriptome analysis based on the highest shikimic acid content single plant 1# and the lowest single shikimic acid content single plant 24# showed that a total of 633 differential genes were detected,256 genes were upregulated relative to 24#,and 377 genes were down-regulated.The pathways that KEGG is most enriched by are "phenylpropane biosynthesis","flavonoid biosynthesis",and "starch and sucrose metabolism".The metabolic pathways related to shikimic acid and downstream metabolism include "phenylpropane biosynthesis","flavonoid biosynthesis","flavonoid and flavonol biosynthesis","phenylalanine metabolism" and "tyrosine metabolism",which further proves that the difference of shikimic acid is strongly correlated with the downstream.(6)Based on the combined analysis of metabolome and transcriptome,the downregulation of the chorismate synthase(CS)gene Gb14157 was one of the main reasons for the accumulation of shikimic acid.Phenylalanine and tryptophan formed feedback inhibition of their respective synthase genes Gb14548 and Gb29155 when the content was high,and it was speculated that Gb14548 and Gb29155 were feedback genes of phenylalanine(Phe.)and tryptophan,respectively,and it was speculated that the cause of tyrosine accumulation was the downregulation of ADT gene(Gb14548)expression,thereby increasing the carbon flow of tyrosine synthesis pathway.The high expression of PAL,CHS,CHI and F3 H genes has obvious positive effects on the accumulation of flavonoids and flavanols,and it is speculated that Gb16672,Gb19001 and Gb07706 may be key genes involved in the positive regulation of ginkgo flavonoids.The differential transcription factors Gb MYB10,Gb MYB23 and Gb MYB41 belonged to the R2R3 MYB family and were all significantly downregulated in shikimic acid single plant 1#.In summary,it is speculated that the accumulation of shikimic acid is due to the downregulation of downstream CS genes and the reduction of amino acid synthesis:the strict feedback regulation mechanism of amino acids is conducive to mutual regulation of metabolic flux balance,and differential transcription factors Gb MYB10,Gb MYB23 and Gb MYB41 may regulate flavonoid pathways while inhibiting shikimic acid synthesis. |
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