| Cyclocarya Paliurus(C.paliurus)is a arbor of Cyclocarya of Juglandaceae with high medicinal value.There is a folk custom of drinking C.paliurus tea,which has the effect of heat-clearing,detumescence and anti-inflammation.The researchs showed that there were active substances of hypoglycemic and anti-hyperlipidemia in the extracts of C.paliurus leaf and these active substances were mainly polysaccharides,flavonoids and terpenoids.In the past,most of the studies focused on these components and pharmacokinetics,but there was no report on the biosynthesis related genes and biosynthesis pathway of these hypoglycemic components.Analysing the molecular mechanism of hypoglycemic biosynthesis of C.paliurus is an important basics for genetic improvement of its medicinal quality.In this study,multi-omics(genome,transcriptome,and metabolome)techniques were used to excavate the related genes of hypoglycemic component from C.paliurus and construct their biosynthesis pathways.The main research contents and results are as follows:(1)Sequencing,Assembly and Annotation of the Genome of C.paliurus: Flow cytometry was used to estimate the genome size of P.cynanomornis(use Zea mays L B73 as an internal reference)and result is about 675 Mb,and the heterozygosity was 3.5%which shows that the heterozygosity of C.paliurus is high in dicotyledonous plants.The genome of C.paliurus was sequenced based on pacbio CCS sequencing and the sequenced data is assembled.The genome size of C.paliurus was about 634.90 MB,N50 reached 34.21 MB and GC content reached 37.35%.The scaffolds of C.paliurus genome were anchored to 16 chromosomes by Hi-C assisted assembly technology,and the loading rate was 95.78%.(2)Comparative genomic analysis of C.paliurus: Phylogenetic tree showed that C.paliurus was closely related to Juglans sigillata,Juglans regia and Carya illinoinensis,and differentiated from Juglandales about 240 million years ago(Mya).It has a common ancestor with J.sigillata,J.regia,C.illinoinensis and Carya cathayensis,and its differentiation time is later than that of Fagaceae[about 680 million years ago(68 Mya)]and Rosales [about 940 million years ago(94 Mya)].The whole genome duplication(WGD)of C.paliurus occurred in 3.22-4.86 Mya.There are 5406 expansion gene families and 4650 contraction gene families in the genome of C.paliurus.Most of the expansion genes are concentrated in signal transduction,transport,catabolism,environmental adaptation and other secondary metabolic biosynthesis;A mass of contraction genes are mainly concentrated in phenylpropane biosynthesis,photosynthesis,oxidative phosphorylation and pyruvate metabolism.The dominant properties and extension of flavonoids and polysaccharides metabolism related genes in C.paliurus may promote the biosynthesis of flavonoids and polysaccharides,so that the content of the metabolite of hypoglycemic increased.Collinearity analysis showsthat some chromosomes of C.paliurus were reversed or fused with J.regia and J.sigillata,such as: Partial inversion between Jrchr12 and Cpchr13,partial inversion between Jschr03 and Cpchr02,partial inversion between Jschr08 and Cpchr07,partial fusion of Jschr02 and Jschr04 into Cpchr09,partial fusion of Jschr01 and Jschr16 into Cpchr09,etc.(3)Metabolome analysis of the changes of hypoglycemic metabolites in C.paliurus:A total of 751 metabolites were detected in the widely targeted metabolome,including 144flavonoids(19.17%),137 phenolic acids(18.24%),81 organic acids(10.79%),69terpenoids(9.18%)and 87 lipids(11.58%).The contents of terpenoids(6,9-dihydroxy-7-macropene-3-one,hawthorn acid,2-hydroxyoleanolic acid,ajianolic acid)in mature and old leaves were higher than those in young leaves and up-regulated significantly.Metabonomic data showed that the differences of metabolites were mainly concentrated in tryptophan metabolism,phenylpropanoid biosynthesis,flavone and flavonol biosynthesis,purine metabolism,pyrimidine metabolism and glycerol phospholipid metabolism.Metabolomics data showed that a total of 751 metabolites were detected.The content of secondary metabolites and enrichment of metabolic pathways in mature and old leaves groups were not significantly different,and were significantly up-regulated compared with young leaves.The hypoglycemic metabolites(terpenoids and polysaccharides)in old leaves groups were significantly higher than those in mature and young leaves groups.(4)Differential expression of related genes of hypoglycemic metabolites in C.paliurus by whole-transcription analysis: A total of 31247 m RNAs,6816 lncrnas,735 circrnas and486 mi RNAs were identified,by pairwise comparison,there were 1405 lnc RNAs differentially expressed(DE)(763 up-regulated,642 down-regulated),8 circ RNAs differentially expressed(4 up-regulated,4 down-regulated),273 mi RNAs differentially expressed(170 up-regulated,103 down-regulated),and 6138 m RNAs differentially expressed(2756 up-regulated,3382 down-regulated).De-circ RNAs are mainly enriched in lipid metabolism,m RNA catabolism,glycosphingomyelin biosynthesis,fatty acid degradation,amino sugar and nucleotide sugar metabolism and RNA degradation.Dem RNAs are mainly enriched in catalytic activity and binding.De-mi RNAs are mainly enriched in monoterpene biosynthesis,mutual transformation of pentose and gluconate,plant hormone signal transduction,plant pathogen interaction and m RNA monitoring pathway.De-lnc RNAs are mainly enriched in plant hormone signal transduction,plant pathogen interaction,starch and sucrose metabolism,endoplasmic reticulum protein processing and endophagy.These results show that these genes related to De-RNAs are significantly enriched in plant hormone signal transduction and the interaction between plants and pathogens.(5)Combined analysis of Whole-transcriptome and metabolome: In the triterpene pathway,we identified 16 important enzyme genes and 3 metabolites.Except for the downregulated expression of Cp IDI and CPSS genes,the expression of most genes(Cp ATAC、Cp PHMGS、Cp MVK、Cp MVD、Cp DXS、Cp CMS、Cp CMK、Cp ISPF)was up-regulated in 10M-OL and 10M-ML groups.In contrast,in the 10M-ML or 10M-OL groupswere found that up-regulated metabolites(mevalonate,1-deoxy-d-xylose-5’-phosphate,Dglyceraldehyde-3-phosphate)are accumulated.The results showed that the DAMs and DEGs which related to triterpene biosynthesis in C.paliurus leaves may be unstable or transcriptional delay at different periods of maturity.15 DEG and 7 DAM were identified which participated in polysaccharide biosynthesis.Most DEGs(Cp SUS、Cp AXS、Cp PGM、Cp GPI、Cp HK、Cp UGDH、Cp RHM)were up-regulated in 10M-ML than in 10M-YL,It leads to an increase in the content of a large number of polysaccharide metabolites(sucrose,fructose,glucose-1-phosphate,glucose-6-phosphate,mannose-6-phosphate and uridine diphosphate glucose).Among the three different leaf age groups,the contents of secondary metabolites in mature and old leaf groups were not significantly different,and were higher than those in young and young leaf groups.Triterpenoids and polysaccharides in mature leaf group were higher than those in old and young leaf groups.Therefore,the mature leaves of Cyclocarya paliurus are the best harvest targets for developing hypoglycemic and anticancer drugs as well as natural sweeteners. |
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