| Resina Draconis,a rare and precious traditional medicine in China,is known as the"holy medicine for promoting blood circulation".It has the functions of promoting blood circulation and removing blood stasis,calming pain and hemostasis,astringent sore and generating muscle.According to the national drug standard,it's derived from the resin extracted from the wood of D.cochinchinensis(Lour.)s.c.chen,a Liliaceae plant,which has been regarded as the second level endangered plant in China since 1991.At present,the extraction raw materials of dragon's blood in China are completely dependent on Laos,Vietnam and other Southeast Asian countries,and with the increasing of demand,the resource of Resina Draconis in foreign countries is also increasingly scarce.A variety of Dracaena plants can form red resin when suffered from physical injury.The pharmacopolist often mixed the resin wood of several Dracaena plant and supplied the medicinal materials market,and some even mixed the red resin wood of other families and genera species.The safety of clinical medication and normal market circulation of Resina Draconis were seriously affected.Therefore,the accurate identification of Resina Draconis is of great significance.However,the research of molecular identification of Resina Draconis is still blank.In addition,according to our survey,there is a kind of Dracaena plant(named Yan-zong)in China,Laos,Vietnam and other Southeast Asian areas,which has a large number of distribution and can form red resin.It is often used as the extraction material of Resina Draconis,However,the quality of its medicinal materials is unknown.In addition,the standard of medicinal materials stipulates that the resin wood of D.cochinchinensis is the genuine extraction material of Resina Draconis.Based on the above,we first evaluated the identification efficiency of traditional DNA barcode fragments for Dracaena.spp,then the chloroplast genome was used to identify the original plant of Resina Draconis.On this basis,the suitable candidate DNA barcodes were selected to study the specific barcode of Dracaena.spp plant.Secondly,the study compared the main chemical components of Resina Draconis which derived from different Dracaena species(D.cochin-chinensis and D.sp),then compared the overall chemical components used metabonomics method in order to evaluate the quality differences.After that,the differences in chemical composition of the healthy stem wood of the original plant were compared using the metabonomics.Finally,the formation mechanism of Resina Draconis was analyzed by high-throughput transcriptome sequencing.The main results are as follows:1.The efficiency of routine DNA barcodes(ITS2,psbA-trnH,rbcL,matk)for identification of Dracaena plants is very low,and among them,the most efficient bar-code sequence is matk,which its efficiency is only 42.86%.The results showed that super-barcode can distinguish Dracaena plants very well using the chloroplast genome as super-barcode.On the basis of chloroplast genome sequencing,the identification effi-ciency of four highly variable fragments(trnP-psaJ,psbK-psbl?trnT-trnL and clpP)was compared.The results showed that only clpP fragments could identify 7 Dracaena species accurately,but the sequence were longer,and the expansion and sequencing success rate were lower."psbK-psbl+trnP-psaJ" can be used for accurate molecular identification of Dracaena plant,and has high success rate of amplification and sequenc-ing.It can be used as the best DNA barcode for molecular identification of dragon's blood and Dracaena.spp.In addition,according to the phylogenetic tree constructed by chloroplast genome and DNA barcode,we thought that D.cochinchinensis and D.cam-bodiana should not be merged into one species,but should be listed separately.2.There were significant differences in the contents of the other four components except for loureirin A.The Resina Draconis derived from D.cochinchinensis contains higher content of resveratrol and 7-4 '-dihydroxyflavone,and the dragon's blood derived from D.sp contains higher content of loureirin B and pterostilbene.The content of loureirin B is higher in the Resina Draconis derived from D.cochinchinensis(9.4 times).the author thinks that Resina Draconis derived from D.sp has the potential to be considered as a substitute of the genuine Resina Draconis.In addition,the content of Loureirin A is much higher than that of Loureirin B(26 times),and both of them are the main active components of Resina Draconis to promote blood circulation and remove blood stasis.Considering that the content of Loureirin B in most of the Resina Draconis material can not meet the National Drug Standard(0.4%),therefore,it is suggested that the relevant departments should further improve the drug standard of dragon's blood,and take the content of Loureirin A or the sum of Loureirin A and B as the content index of Resina Draconis quality control.3.A metabonomics method based on UHPLC-Q-Orbitrap-MS was established to study the difference of the chemical composition of Resina Draconis derived from D.cochinchinensis and D.sp.The OPLS-DA analysis results showed that there were significant differences in the chemical composition of Resina Draconis of different sources.Finally,a total of 154 different compounds were identified(or temporarily identified),including 93 compounds with high content in the Resina Draconis derived from D.sp,and 61 compounds with high content in Resina Draconis derived from D.cochinchinensis.The results showed that the contents of amines,phenolic acids,alkaloids,terpenes,organic acids and steroids were higher in Resina Draconis derived from D.sp,while the contents of phenylpropanoids and flavonoids were higher in Resina Draconis derived from D.cochinchinensis.In addition,eight compounds were selected as the differential markers to distinguish the two sources of Resina Draconis.4.Comparing the chemical composition difference of healthy stem wood of D.cochinchinensis and D.sp using the metabonomics.The results of OPLS-DA analysis showed that there were significant differences in chemical composition between the two species(R2Y=0.998,Q2=0.954).Among the 21,955 features analyzed,there are 619 that meet the requirements of VIP>1.0 and P<0.01(t-test),which is 470 less than the difference compounds after injury induction,which indicated that there was a certain difference in the chemical composition of the stem wood of D.cochinchinensis and D.sp before injury induction,and this difference becomes more and more obvious after injury induction.Finally,we identified(or temporarily identified)79 compounds.The results of normalized peak area analysis showed that there were higher amino acids and organic acids in the healthy stem wood of D.cochinchinensis,and higher flavonoids,alkaloids,steroids and amines in the healthy stem wood of D.sp.5.We sequenced the transcriptome of the stem samples of D.cochinchinensis after injury induction.The results showed that the number of Unigene was 63,244,and 27,912 Unigenes were annotated.The digital expression patterns of different induction time points were constructed and verified by qPCR.The statistical results of differentially expressed genes showed that the first three days after injury induction was the most active period of stress response.The results of GO term enrichment and KEGG pathway showed that the physiological processes related to growth and development,organ development and reproductive system development were in the down-regulation stage within three days after injury induction,while the processing of various secondary metabolites biosynthesis,photosynthesis and stress resistance were in the up-regulation stage.However,the up-regulated processing of biosynthesis pathway and stress resis-tance pathway was gradually down-regulated in three days after injury induction.The metabolic pathways related to flavonoids(the main active components of dragon's blood)were not significantly enriched by differentially expressed genes at first three days of injury induction,but multiple differentially expressed genes were enriched to related pathways after three days of injury induction. |
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