| Purpose:This research is part of the National Natural Science Foundation of China's "Study on the accumulation of arctiin in the development process of Arctii Fructus based on frozen section-microdissection-liquid mass spectrometry(81773852)".In order to understand the dynamic accumulation of chemical components during the growth and development of A.lappa,this study determined the content of arctiin and arctigenin in five different growth stages and dried A.lappa fruits,and different germination stages of A.lappa.Identification and comparison of the chemical components in the fruits and seeds of A.lappa at five different growth stages;the purpose is to clarify the dynamic accumulation of chemical components during the growth and development of A.lappa,to provide the biosynthesis mechanism of the chemical components such as arctiin in A.lappa fruit,The determination of the harvest date and the research on the plant protection of A.lappa provide theoretical references.Transcriptome sequencing and analysis of different organs of the A.lappa plant,combined with the accumulation characteristics of arctiin and arctigenin content in the A.lappa fruit at different growth stages and the gene data of arctidium,the biosynthesis-related candidate genes of arctiin and arctigenin in A.lappa.The analysis will provide references for the biosynthesis research of arctiin and arctigenin,and provide valuable resources for the molecular biology and genetic function analysis of arctium.Materials and Methods:1 Material: Take five different growth stages(flower bud period,first flower period,full bloom period,end flower period,maturity period)A.lappa fruit,and take the dried and mature A.lappa fruit harvested in the same year.All A.lappa fruits were collected in September 2018 from June to September 2019 from the Dalian campus of Liaoning University of Traditional Chinese Medicine(Key species preservation nursery for medicinal plants in Liaoning Province,N39°06?18?,E121°87?62?).2 Methods:2.1 By consulting related materials and websites such as Flora of China,the Plant List,etc.,the classification of Arctium plants is sorted,and the related biological activities,chemical diversity and molecular biology of Arctium plants are summarized.2.2 High performance liquid chromatography(HPLC)was used to determine the content of A.lappa fruit at different growth stages and A.lappa germination stages to understand the dynamic accumulation of arctiin and arctigenin components during the growth and development of A.lappa.2.3 The experiment uses ultra high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry(UPLC-Q-TOF-MS)to identify the chemical components in the fruits and seeds of A.lappa at five different growth stages,and compare the selection of each compound in different samples The ionic response intensity can achieve the two research goals of the structure speculation and relative content difference of the main effective components in the fruits and kernels of A.lappa at five different growth periods.2.4 RNA extraction,library construction,transcriptome sequencing and assembly were performed on the bud stage fruits,the first flowering stage fruits,the full bloom stage fruits,the late flowering stage fruits,the mature stage fruits,leaves,stems,perianths,petioles and roots of the same plant of A.lappa.Compare Unigene with NR,NT,Swiss-Prot,KEGG,COG,GO by blastx,and count the number of unigenes annotated to each library and all annotations respectively.Use the assembled unigenes as a reference sequence,and find all SSRs through the SSR software Micro SAtellite(MISA).At the same time,the transcriptome difference analysis of different organs of A.lappa is also carried out to understand the biological function of the different genes.2.5 Summarize the research on the biosynthesis pathway of lignans,and screen the related enzymes for the biosynthesis of arctiin.Use Arabidopsis protein sequence and other compositae plants to blast comparison of A.lappa genomic protein files,and use Pfam?scan to search whether the protein contains the corresponding domain.The correlation analysis of 12 related enzyme genes in arctiin and arctigenin biosynthesis pathways in A.lappa was carried out.Results:1 At present,a variety of compounds have been isolated from A.lappa plants,including lignans,terpenoids,flavonoids,phenols,lactones,alkynes and polysaccharides,etc.These compounds are distributed in different plant parts,among which lignans It is the most characteristic component in the genus Arctium.A.lappa,as the most commonly used medicinal plant in the genus Arctium,has antipyretic,antibacterial,diuretic,hypoglycemic,antioxidant,anti-inflammatory,anti-hepatotoxic,anti-ulcer and anti-tumor activities and other pharmacological activities.2 In the first three growth periods,arctiin and arctigenin accumulate in a small amount.At the end of the flower,a large amount of arctigenin and a small amount of arctiin are produced.At this time arctigenin is the main component;while in the mature period,the A.lappa fruit a large amount of glycosides accumulate,and the content of arctigenin decreases.At this time,arctiin is the main component and the content is far greater than arctigenin.During the germination process of A.lappa,the arctiin content decreases with time,and the arctiin content increases.At 15 days of germination,the arctiin content is higher than arctiin.3 A total of 31 compounds were identified from five samples of A.lappa fruit at different growth stages.The identified components are classified according to structural types,including amino acids(2),flavonoids(1),and phenolic acids(7,including 6 benzenes).Propyl phenolic acid)and lignans(21).Among them,20 compounds were identified in the bud stage fruits,the same 17 compounds were identified in the initial and full-flowering stage fruits,and 28 compounds were identified in the final flowering stage fruits.The A.lappa fruit at the mature stage contains the largest variety of components,including all 31 compounds.A total of 33 main chromatographic peaks were observed in A.lappa kernels at five different growth stages,and 32 possible structures were identified.The identified components include amino acids(1),phenolic acids(8),lignans(19),and fatty acids(4);among them,26 are identified in the initial flowering and bud stage kernels.Among these compounds,29 compounds were identified in the seeds at the full-flowering stage,and 32 compounds were identified in the seeds at the end of the flowering stage.The species of A.lappa seeds in the mature stage are the most diverse,including all 33 compounds.4 Using the Illumina Hi Seq TM 2500 sequencing platform to sequence the A.lappa transcriptome,474 950 882 clean reads were obtained,and a total of 161 596 unigenes were generated from de novo assembly.A total of 100,834 annotated unigenes were obtained.The number of annotated unigenes in the NR,NT,Swiss-Prot,KEGG,COG,and GO databases were 93 853(58.08%),77 118(47.72%),64 270(39.77%).,60 057(37.16%),43 841(27.13%),70 477(43.61%).It is annotated to 56 branches of the three GO categories of biological process,cell composition,and molecular function.Using public databases for homology comparison,60 057 unigenes were annotated into 128 metabolic pathways,7 180 genes were annotated to the biosynthesis of secondary metabolites,of which 387 genes were involved in phenylalanine metabolism and 868 genes Participated in the biosynthesis of phenylpropane.The total number of identified SSRs was 33 002,and the number of SSR-containing sequences was 26 254,of which 5252 unigenes contained more than one SSR site.The frequency of SSR loci was 20.4%.5 Compared with A.lappa,8 4CL genes,2 C4 H genes,and 6 CCo AOMT genes,2CCR genes,5 PAL genes,2 C3 H genes,8 CAD genes,5 COMT genes,3 DIR genes,5 HCT genes,3 PLR genes,6 SIRD genes.Pearson correlation coefficient was calculated based on arctiin content and related gene expression(cor>0.7,P<0.05).The results showed that a 4CL(ID: evm.model.000137 F.68)gene,a DIR(ID:evm.model.000171 F.169)gene and a HCT(ID: evm.model.000054 F.303)gene are associated with arctiin The composition is highly correlated.Conclusion:1 The related biological activities,chemical diversity and molecular biology of Arctium are summarized.The research provides a basis for the further development and rational application of the pharmacological activity,clinical application,biosynthesis and metabolism of Arctium.2 Arctigenin is the main chemical component in the A.lappa fruit at the end of the flower,and its content is higher than arctiin.Arctiin is the main chemical component in the A.lappa fruit at the mature stage and its content is as high as 11.72%,and the content of arctigenin is significantly reduced compared with the end of the flowering stage.As the A.lappa grows,the content of arctiin and arctigenin changes,and it can be inferred that the arctigenin will be converted into arctiin as the A.lappa matures.The arctiin content in the dried A.lappa fruit harvested in the same year was 8.591%,which is higher than the 2020 version of the "Chinese Pharmacopoeia" that the arctiin content should not be less than 5%.With the increase of germination time,the content of arctiin decreases,and the content of arctigenin increases.At 15 days of germination,the content of arctigenin was greater than that of arctiin.The results are completely opposite to the accumulation of A.lappa fruit during the growth period,indicating that the A.lappa plant undergoes at least two transformations from arctigenin to arctiin from germination to development.3 Using UPLC-Q-TOF-MS technology,five different growth stages of A.lappa fruit and seed kernels were used to identify compounds.As a result,31 components were obtained,including amino acids,flavonoids,phenolic acids and lignans.A total of 33 main chromatographic peaks were observed in five different growing A.lappa seeds,and 32 possible structures were identified,mainly amino acids,phenolic acids(caffeic acid derivatives such as chlorogenic acid),lignans and fatty acids Ingredients.Among them,arctiin and arctigenin,as the main pharmacological components and quality marker components of A.lappa seeds,were identified in the fruits and seeds of five different periods.4 The use of transcriptome sequencing technology to obtain A.lappa transcriptome data lays a foundation for the identification of the function of A.lappa genes,the analysis of secondary metabolic pathways and the research of its regulatory mechanism.The analysis of the simple repetitive sequence(SSR)locus information lays the foundation for the development of molecular markers.5 By summarizing the research on the biosynthetic pathways of lignans,the possible biosynthetic pathways of arctiin and arctigenin in A.lappa fruit are obtained;the related enzyme genes that may be related to the metabolism in the biosynthetic pathway of arctiin and arctiin in A.lappa fruit are related In the sex analysis,a 4CL gene,a DIR gene and HCT gene are highly correlated with arctiin biosynthesis.These data provide references for studying the synthetic pathways of arctiin and arctigenin,and provide valuable resources for the biological research of arctium. |
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