Based On Gene Sequencing And UPLC-Q-TOF/MS On Four Callerya Species Chloroplast Genome Characteristics Analysis And Chemical Composition Analysis Were Performed

Callerya reticulata(Bentham)Schot、Callerya dielsiana(Harms)P.K.Loc ex Z.Wei & Pedley、Callerya nitida var.hirsutissima(Z.Wei)X.Y.Zhu and Callerya nitida(Bentham)R.Geesink are traditional Chinese medicinal materials,with the effect of activating blood and replenishing blood,relaxing meridians and activating the nerves,etc.Due to its high similarity in appearance,it is more confusing in folk and market circulation.The current research on the medicinal plants of Callerya is mainly focused on the chemical composition of Callerya nitida var.hirsutissima,and the research on Callerya reticulata,Callerya dielsiana and Callerya nitida is weak in this aspect,and there are few reports on the molecular biology of these four medicinal plants.In this study,we used chloroplast genome sequencing,liquid mass spectrometry and other analytical techniques,combined with data mining,to systematically analyze and compare these four Callerya medicinal plants in terms of appearance,microscopy and thin layer,molecular biology and chemical composition,to study the chloroplast genome and chemical composition of the four and analyze the differences at different levels to provide data support for the standardization of drug use.PurposeIn this study,a resource census of Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida was conducted and compared in appearance,microscopic and thin-layer experiments,with a view to providing a reference for the identification of the four medicinal plants.We used chloroplast genome sequencing and liquid chromatography to construct a phylogenetic tree at the molecular and chemical levels to analyze the relatives of the chloroplast genome,to mine the chloroplast DNA barcodes with high specificity for the species,and to compare the chemical composition of the four Callerya medicinal plants and screen the differential compounds,so as to lay the foundation for in-depth research on the chemical composition and molecular identification of the four Callerya medicinal plants.In order to clarify the differences in chemical composition and affinities of Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida,this study was conducted to systematically compare and analyze these four Callerya medicinal plants in terms of appearance,microscopic and thin-layer experiments,molecular biology and chemical composition,using analytical techniques such as chloroplast genome sequencing and liquid mass spectrometry to study the differences between the four at different levels for rational and standardized use.Methods1.Experimental samples of Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida were collected through the Plant Species Information System to compare the appearance,microscopy and thin-layer experiments of the four herbs.2、Fresh leaves of Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida were collected and DNA was extracted according to the plant genomic DNA extraction kit from Tiangen Biochemical Technology Co.,and chloroplast genome sequencing was sequenced by second-generation sequencing and third-generation sequencing technology.The chloroplast genome was assembled using bioinformatics analysis software,and the chloroplast genome was annotated using the software combined with the NCBI database,and the complete chloroplast genome spectrum was made using the software.A series of Bioinformatics analysis software was used to perform codon preference analysis,simple repeat analysis,accounting diversity analysis,homologous collinearity analysis and phylogenetic tree analysis of chloroplast genome.3、Data were collected based on UPLC-Q-TOF/MS technique for Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida alcohol extracts to obtain precise molecular weights,peak intensities and secondary mass spectra numbers.The mass spectrometry data of the control solution were combined with the literature to infer the cleavage pattern and deduce the structure of the compounds,and the metabolomics method,combined with multivariate statistical analysis,was applied to screen out the differential compounds.Result1、The wild-collected herbs were mainly in the areas of Fengcheng City and Yichun City,Jiangxi Province,with the most abundant resources of Callerya reticulata,followed by Callerya nitida var.hirsutissima and Callerya nitida,and the more scarce resources of Callerya dielsiana.Microscopic experiments revealed some differences in the cell molecules and inclusions of the four herbs,and the results of thin-layer experiments indicated that all four herbs contained formononetin.2 、 Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima,and Callerya nitida had 193,204,236 and 202 simple repeat sequences,i.e.,SSR loci,respectively.the strongest preference for Callerya reticulata was for the codon AUG encoding methionine.Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida had the strongest preference for codon UUA,which encodes leucine(Leu),and all four species had Chloroplast genome codon preferences ending in A/U(T)bases.The chloroplast genomes of Callery nitida var.hirsutissima,Callerya nitida,Callerya dielsiana and Callerya reticulata share the same homologous fragments,and Callerya nitida var.hirsutissima r RNA is rearranged compared to the other three species.Nucleic acid diversity analysis showed that the PI value in the RPL32 region was the highest.The phylogenetic tree was divided into six major branches,in which Callery nitida var.hirsutissima,Callerya nitida,Callerya dielsiana and Callerya reticulata clustered as one,Callerya nitida var.hirsutissima and Callerya dielsiana are divided into different branches.3、UPLC-Q-TOF-MS technology was used for medicinal plants Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida for chemical composition analysis,A total of 52 compounds were presumed to be identified in Callerya reticulata extract,48 compounds were presumed to be identified in Callerya dielsiana extract,93 compounds were identified in Callery nitida var.hirsutissima extract,and 79 compounds were identified in Callery nitida extract.Mainly flavonoids,a small number of organic acid compounds and other compounds.The four species have a common composition of 35,including 24 flavonoids and 11 other compounds.The data such as peak intensity and retention time were imported into SIMCA14.1 software to screen the differential compounds,the results were as follows,A total of 17 different compounds were identified between Callerya reticulata and Callerya dielsiana,A total of 33 different compounds were identified between Callerya reticulata and Callerya nitida var.hirsutissima,33 different compounds were identified between Callerya reticulata and Callerya nitida,32 different compounds were identified between Callerya dielsiana and Callerya nitida var.hirsutissima,32 different compounds were identified between Callerya dielsiana and Callerya nitida,26 different compounds were identified between Callerya nitida var.hirsutissima and Callerya nitida.The four Callerya species have a total chemical composition of 35.ConclusionIn this study,the four Callerya herbs and fresh plant leaves were collected in the field to compare the appearance and traits of the four herbs,and DNA samples meeting the sequencing requirements were extracted to sequence and characterize the chloroplast genomes of the four species simultaneously for the first time.The comparison and analysis of the sequencing results revealed that the four Callerya medicinal plants differed in chloroplast genome length,gene function,codon preference and gene order,respectively,and the rpl32 fragment,as a highly variable region.In the comparison of the data obtained from UPLC-Q-TOF/MS analysis,it was found that all four herbs differed significantly in the type of chemical components.In this study,four medicinal plants of Callerya reticulata,Callerya dielsiana,Callerya nitida var.hirsutissima and Callerya nitida were compared at the molecular and chemical levels to lay the foundation for the study of chemical composition,affinities and future molecular identification of these four Callerya medicinal plants.