| Sinopodophyllum hexandrum(Royle)Ying,family Berberidaceae,the only species of this genus in China,is a kind of rare and endangered Chinese medicinal materials.The root and rhizome of S.hexandrum riches in podophyllotoxin that is cancer drugs precursors to synthesis VP-16(etoposide),VM-26(teniposide),GP7 and NK6 ll.It has attracted much attention because of the anticancer activity.In the field of biology,the researches on S.hexandrum have become the hotspot and focus.S.hexandrum is widely distributed and a dramatic difference of regional environment may lead to the difference chemical composition,phenotypic traits and genetic diversity,finally,lead to the quality differences.As the great medicinal value in S.hexandrum is gradually found,much more studies on it increased,among them mainly focused on the extraction,separation and determination for chemical composition and some biological functions,however,the research on chemical diversity of S.hexandrumis comparatively few.Therefore,on the basis of resource survey,this work comprehensively studied the relationship between S.hexandrum's chemical diversity and phenotypic diversity,genetic diversity and the environmental factors by chemometrics methods and quantitativeecology methods.Through sequenced and analysis for transcriptome of S.hexandrum,the study determined the effect of main influence factors on its chemical composition,evaluated its quality,revealed its best growing region,and knew about the key genes to control the synthesis of podophyllotoxin.Moreover,this text presented some protection suggestions for S.hexandrum,which are important for selection the excellent varieties,new drug development,implementation the cultivation of medicinal material,and providing basis for further study on S.hexandrum's gene function.Finally,in order to ensure the safe and effective of clinical application,this study promoted the sustainable utilization of the resources of Chinese medicine,protected the development of modern Chinese medicine industry.The main conclusions are as follows:1.A stable fingerprint determination method was established and it indicated that chemical composition content had larger differences in different geographical region and showed abundant chemical diversity.In addition,the quality of S.hexandrum from different regions was evaluated by stoichiometry coupled with chromatographic data,such as multivariate analysis.Morphological and visual classification,hierarchical cluster analysis,principal component analysis and discriminant analysis results consistently showed that eight group S.hexandrum collection from different areas was classified into three groups and the quality of S.hexandrum in Yongdeng county,in Gansu province(S4),was the best.2.The study showed that the phenotypic character of S.hexandrum are abundant variation and significant difference(P< 0.05)between phenotypic traits(regional survey)and group(regional survey)through researching30 phenotypic traits of 240 samples from 8 different areas.The differentiation of individuals in the same investigation area was larger than that of among different investigation regions,however,the phenotypic traits stability of individuals in the same investigation area was lower than that of among different investigation region.The phenotypic traits of reproductive organs with more differentiation and poorer stability lower compared to vegetative organs(VST: plant height(25.827%)> flower(19.376%)> fruit(16.248%)> leaf(14.914%)).Linear regression analysis showed that there was no significant correlation between phenotypic diversity and its chemical diversity(P< 0.05),which indicated that the chemical diversity of S.hexandrum was not determined by phenotypic differences and species of S.hexandrum may produce different chemical types.3.11 primers of ISSR amplification 263 clear and stable stripe showed that S.hexandrum had a low genetic diversity,whose polymorphism rate was 55.89% and He averages value was 0.0585.UPGMA,PCo A and Bayeian clustering analysis consistent showed that 32 S.hexandrum populations were divided into 3 clusters withclear genetic structure.The significant genetic variation occurd among S.hexandrum by AMOVA analysis(63.77%,P< 0.0002)and Gststatistics(Gst = 0.3623).Mantel test showed that the correlation of spatial pattern and geographical location of S.hexandrum is not significant(r = 0.213,P = 0.213)(P< 0.05).High genetic differentiation of S.hexandrum may be related to the limited gene flow(Nm=0.8801)and limited distance of seed propagation.The results indicated that the correlation between genetic diversity and chemical diversity of S.hexandrum is not significant(P< 0.05)and its chemical diversity is not affected by the genetic diversity by Linear Regression Analysis.Considering the lower genetic diversity of S.hexandrum,higher genetic differentiation and abundant chemical diversity and the large pressure from human,situ protection was the best choice,and ex situ conservation was the supplement measure.4.Environmental factors significantly affected the geographical differences of S.hexandrum's chemical composition and contributed to its chemical diversity.The main environmental factors affecting the chemical diversity included the average annual rainfall,Julymean temperature,frost-free period,sunlight hours,p H,organic matter and available potassium,among them,the first four factors are the deciding factors,and the average annual rainfall was the most important factor that showed a significantly negative correlation with the content of chemical composition(P< 0.05).Organic matter,p H and available potassium were the limiting factors.The content of organic matter played the most important limiting that was significantly positive with chemical composition(P< 0.05).Considered from chemical component contents,Yongdeng county in Gansu province(S4)was best production location to product S.hexandrum with high content podophyllotoxin and lignans.Shangri-la county in Yunnan province(S6)and Nyingchi county in Tibet(S7)were suitable to product S.hexandrum that rich in high content of quercetin and kaempferol.5.The transcriptome sequencing obtained 1634670 high quality sequence.Among them,74026 had high credibility Unigenes by assembly and functional annotation.34175 Unigenes can directly determine its CDS area and the direction of the sequence.34175 Unigenes can be directly determined its CDS and the sequence direction by Nr,Swiss-Prot,KEGG,blastx compration in COG protein database(e value < 0.00001)and forecasting for coding region based on ESTScansoftware.The number of Unigenes comparison to the wild strawberry(Fragaria vesca),cocoa(Theobroma cacao)and grape(Vitis vinifera)were large,23.16%,14.61%,4.88%,respectively.14885 terms were classified into 69 functional category through the GO function classification.The number of genes categorized into metabolic and cell function were large,according to 21.62% and 10.34% of the total number of Unigenes.Through the COG classification,21604 Unigenes were classified into 24 functional category,among them,the number of genes categorized into prediction of general function(R),posttranslational modification,protein turnover,protein chaperones(O)and the ribosome structure and biogenesis were large,accounted for 17.21%,9.74% and 7.87% of the total Unigene.11071 Unigenes were obtained in 125 major metabolic pathways,including biosynthetic pathway of phenylpropanoids that is related to the synthesis of podophyllotoxin and synthetic metabolic pathways of flavone and flavonol that is related to the synthesis of flavone.6.The result founded 14 enzyme genes releated to the synthesis of podophyllotoxin that include 11 cinnamyl alcohol dehydrogenase(CAD),2pinoresinol-lariciresinol reductase(PLR)and 1 Dirigent protein.Research on above genes and transcription could reveal the mechanism of synthesis for podophyllotoxin. |
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