Transcriptomic And Metabolomic Analysis Reveals The Biosynthesis And Distribution Of Active Ingredients In Nanhaia Speciosa

ObjectiveNanhaia speciosa,also known as ’niudali’,has been widely used not only as a traditional food but also a herb in south China.As a perennial woody climber,the root of N.speciosa is nutritious and attracts great attention,which might lead to the waste of the stem and leaf in a large scale.According to previous reports,flavonoids,isoflavones,polysaccharides and amino acids are groups of active compound isolated and identified from N.speciosa.However,there is no record about the research on the biosynthesis and regulation of the above compositions of N.speciosa.This article aims to study the biosynthesis and accumulation of active compounds in N.speciosa utilizing transcriptomic and metabolomic analysis,which might provide basic data for integrated exploitation and utilization of N.speciosa.MethodsThe analysis of flavonol glycosides from N.speciosa was completed through LC-MS;Concentration of flavonoid and polysaccharide in different organs of N.speciosa were measured by UV-spectrophotometry;Concentration of flavonol glycosides,isoflavones and amino acids are confirmed by HPLC;Comparative transcriptomic analysis was applied to reveal structural genes involves in the biosynthesis of flavonoid,isoflavone,polysaccharide and amino acid.Correlation analysis was applied for explain the organic distribution of the above compounds in N.speciosa.Analysis of conserved motif and construction of phylogenetic tree were used for searching for MYB and bHLH transcription factors involved in the biosynthesis of flavonoid and polysaccharide of N.speciosa.Candidate gene encoding key enzymes catalyzing biosynthesis of active compounds from N.speciosa were cloned and prokaryotic expressed,whose in vitro catalyzing activity and enzymatic kinetics were also studied.MspR2R3-MYB26 might modulate the flavonoid biosynthesis of N.speciosa.Results1.Isoquercitrin and isorhamnetin-3-o-glucoside are identified with LC-MS.The concentration of both compounds is highest in leaf and lowest in root;2.Flavonoid contents of leaf and stem were close,and significantly higher than that of root;Concentration of maackiain,formononetin,polysaccharide and amino acid were higher in the root;3.Transcription data from the root,stem and leaf of N.speciosa is generated by RNA-seq.14 PAL(4 DEGs)and 32 4CL(7 DEGs)from the upstream pathway of flavonoid biosynthesis were mined;77 unigenes of structural genes involve in flavonoid biosynthesis were discovered,28 of which are DEGs;Besides,2,20,20 DEGs were found in the pathways of isoflavone(7 unigenes),polysaccharide(218 unigenes),and amino acid(261 unigenes)biosynthesis,respectively;The correlation analysis showed that the active compounds mentioned above might be transported and accumulated in other organs after being synthesized.4.29 R2R3-MYB and 98 bHLH are found by gene mining.The MspR2R3-MYB26 with the bHLH-interacting conserved motif[DE]Lx2[RK]x3Lx6Lx3R,was assigned to subgroup 6 in the R2R3-MYB family and might modulate the flavonoid biosynthesis of N.speciosa.The MspbHLH14,MspbHLH19,MspbHLH20,MspbHLH52,MspbHLH77,MspbHLH78,MspbHLH87 were assigned to subgroup Ⅲ(d+e);MspbHLH80,MspbHLH30,MspbHLH69 belonged to subgroup Ⅲf;and MspbHLH92,MspbHLH41,MspbHLH29,MspbHLH85,MspbHLH79,MspbHLH1,MspbHLH5 were assigned to subgroup VII(a+b),might be involed in the modulation of flavonoid biosynthesis.5.Cloned and prokaryotic expressed MspCHI1 and MspCHI2.In vitro catalyzing experiments verified MspCHI1 as type Ⅱ CHI and MspCHI2 as type I CHI;The Km and Vmax of recombinant protein MspCHI1 on isoliquiritigenin was 49.82 μM and 117.21 nmol min-1,respectively;The Km and Vmax of recombinant protein MspCHI1 on naringenin chalcone was 32.54 μM and 279 nmol·min-1,respectively;The Km and Vmax of recombinant protein MspCHI3 on naringenin chalcone was 32.95 μM and 131.43 nmol·min-1,respectively.Conclusion1.As marker components of Nanhaia speciosa,the content of maackiain,formononetin and polysaccharides are higher in the root of N.speciosa,which supports the traditional utilization of the herb.Besides,the content of total flavonoids in the stem and leaf are significantly higher than that of root,suggests that these parts might has potential to be developed as new resoures to increase added value of N.speciosa.2.Flavonoids could be synthesized in the root of N.speciosa,then transported to other organs.CHI3 and multiple CHS are highly expressed in root,and the two isoflavons-maackiain and formononetin-are also enriched in the same location,indicating that these genes might be involved in flavonoid synthesis in N.speciosa.As for the biosynthesis of polysaccharide,the process may start from photosynthate being transported to stem,then catalyzed to NDP,connected into polysaccharides by GT,and finally stored in root;MspMYB26 contains the motif necessary for the combination with bHLH,which might enable it to regulate the flavonoid biosynthesis in N.speciosa;The candidate bHLH transcription factors involve in the regulation of flavonoid biosynthese in N.speciosa are assigned to subgroup Ⅲ(d+e),Ⅲf,and Ⅶ(a+b).3.MspCHI1 belongs to type Ⅱ CHI while MspCHI2 belongs to type I CHI.With the ability of utilizing isoliquiritigenin as substrate,MspCHI1 may participates in the biosynthesis of isoflavone of N.speciosa.