Identification Of Aroma Components And Synthetic Genes Of Dendrobium Loddigesii Rolfe

Dendrobium loddigesii Rolfe is a perennial epiphytic herb of the Orchidaceae genus Dendrobium.Flower fragrance is one of the important ornamental characters of D.loddigesii.However,the volatile components and biosynthetic process of flower fragrance are very complex,and the flower fragrance is in a dynamic process,which makes the research on medicinal active components,flower pattern and color of D.loddigesii deeper than that of flower fragrance.Therefore,it is necessary to study floral metabolites and genes related to floral biosynthesis of D.loddigesii.In this study,the flowers of high-scent D.loddigesii in Xingyi,Guizhou Province and ligh-scent D.loddigesii in Luofu Mountain,Guangdong Province were used as materials to analyze non-targeted metabonomics and screen differential metabolites and metabolic pathways;Transcriptome sequencing was carried out,and differentially expressed genes related to floral biosynthesis of D.loddigesii were preliminarily screened,and the expression amount was verified;Metabolomics and transcriptomes were combined to analyze the expression of the differential genes obtained from the combined analysis.The flowers of high-scent D.loddigesii were used as materials in bud stage,half-opening stage,blooming stage and declining stage.It is helpful to explore the molecular mechanism of aroma biosynthesis of D.loddigesii and make preliminary preparations for breeding new aromatic Dendrobium varieties.1.In this study,80 compounds,including 11 categories,were identified from the volatile components of flowers of two kinds of fragrant D.loddigesii in full bloom.There are more metabolites of aldehydes（15）,terpenoids（14）and alkanes（11）,and the relative contents of terpenoids,aldehydes and esters are 23.54%,18.03%and 17.71%respectively.PCA analysis and OPLS-DA analysis showed that the metabolites of two kinds of D.loddigesii were obviously separated,and 38metabolites with significant differences were screened out terpenoids（21.85%）,esters（16.69%）and alcohols（8.68%）are the main differential metabolites of high-scent,while heterocyclic compounds（6.84%）,terpenoids（6.43%）and aldehydes（6.06%）are the main differential metabolites of ligh-scent.The metabolites with the greatest difference were geraniol and 4-propyl-phenol,and the difference multiples of Log₂（fold change）were 21.58 and-17.29.2.There are 271 241 852 raw reads in the transcriptome of D.loddigesii,and257 937 448 clean reads are obtained by screening.The percentage of Q20 bases in the transcriptome is greater than 97%,the percentage of Q30 bases is not less than92.95%,the overall sequencing error rate is 0.03%,and the GC content is distributed in 45.1%～46.1%.A total of 175 089 unigenes were obtained,with an average length of 1 168 bp,N50 of 1 745 bp,N90 of 371 bp,total length of 204 522 529 bp,and the length concentrated in 200～2 000 bp,among which 100 264 were the most.69.16%（121 096 unigenes）of Unigenes were annotated in the database,and the largest number of Unigenes were annotated in NR database（120 597 unigenes,68.88%）.Unigene mainly notes three species:Dendrobium candidum,Phalaenopsis equestris and Apostasia shenzhenica.3.Using c DNA of stem,leaf and flower of D.loddigesii as template,the expression stability of six candidate internal reference genes,TUA,TUB,EF-1α-1,EF-1α-2,UBI and NADPH,was analyzed by Ge Norm software,and the result was NADPH=UBI>TUB>EF-1α-2>EF-1α-1.Comprehensively analyzing the results of ge Norm and Norm Finder software,UBI was finally selected as the internal reference gene of D.loddigesii.4.Seventeen structural genes related to floral synthesis of D.loddigesii were screened preliminarily,and their expression levels were verified.The results showed that the change trend of gene expression levels was basically consistent with FPKM.Compared with the light-scented D.loddigesii,the expressions of ECHS,UGT2,GGPPS,TKPR,UQCRFS,PER,MTDH,SDR,MFP-α,KAT were up-regulated,while the expressions of FACE,PAL,C4H,CAD,UGT1,AOC and LOX were down-regulated.5.Combined analysis of metabolomics and transcriptome showed that CAD,PAL and C4H were related to eugenol,LOX were related to（Z）-3-hexenal.The expression level analysis found that in the four flowering stages of high-scent D.loddigesii,the expression level of CAD was the highest in the blooming period,and the expression level was the lowest in the decay period;the expression level of PAL was the lowest in the blooming period,and the expression level was higher and expressed in the other three flowering periods and the level is close;C4H has the highest expression in the bud stage and the lowest in the blooming stage;the expression level of LOX was the highest in the bud period and the lowest in the blooming period.