Advances in research methods for biosynthetic pathway analysis of active ingredients in traditional Chinese medicine / 中国中药杂志

The active ingredients in traditional Chinese medicine(TCM)are the foundation for the efficiency of TCM and the key to the formation of Dao-di herbs. It is of great significance to study the biosynthesis and regulation mechanisms of these active ingredients for analyzing the formation mechanism of Daodi herbs and providing components for the production of active ingredients in TCM by synthetic biology. With the advancements in omics technology, molecular biology, synthetic biology, artificial intelligence, etc., the analysis of biosynthetic pathways for active ingredients in TCM is rapidly progressing. New methods and technologies have promoted the analysis of the synthetic pathways of active ingredients in TCM and have also made this area a hot topic in molecular pharmacognosy. Many researchers have made significant progress in analyzing the biosynthetic pathways of active ingredients in TCM such as Panax ginseng, Salvia miltiorrhiza, Glycyrrhiza uralensis, and Tripterygium wilfordii. This paper systematically reviewed current research me-thods for analyzing the biosynthetic functional genes of active ingredients in TCM, elaborated the mining of gene elements based on multiomics technology and the verification of gene functions in plants in vitro and in vivo with candidate genes as objects. Additionally, the paper summarized new technologies and methods that have emerged in recent years, such as high-throughput screening, molecular probes, genome-wide association studies, cell-free systems, and computer simulation screening to provide a comprehensive reference for the analysis of the biosynthetic pathways of active ingredients in TCM.

Transcriptome-wide identification and expression pattern analysis for Dof gene family in Panax ginseng from Jilin / 中国中药杂志

Dof(DNA binding with one finger), a unique class of transcription factors in plants, play an important role in seed development, tissue differentiation, and metabolic regulation. To identify the number and function of Dof gene family members in Panax ginseng, this study identified the members of Dof gene family in P. ginseng and systematically analyzed their structures, evolution, functional differentiation, expression patterns, and interactions using bioinformatics methods at the transcriptome level. At the same time, the association analysis of Dof genes from P. ginseng with key enzyme genes for ginsenoside synthesis was carried out to screen the candidate PgDof genes involved in the regulation of ginsenoside biosynthesis. The results showed that there were 54 genes belonging to the Dof gene family in P. ginseng from Jilin. All PgDof genes had Zf-Dof conserved motifs, implying that they were evolutionarily conserved and could be divided into five groups. Expression pattern analysis confirmed that the expression of PgDof gene family members in different tissues, different year-old P. ginseng, and different farm varieties varied significantly. Simultaneously, as revealed by "gene-saponin content" and "gene-gene" linkage analysis, an important candidate PgDof14-1 gene involved in the regulation of ginsenoside biosynthesis was obtained. From the established genetic transformation system of this gene in the hairy roots of P. ginseng, a positive hairy root clone was determined. This study has laid a theoretical foundation for the study of Dof gene family in P. ginseng.

Germacrene-derived sesquiterpene lactones:opportunities and challenges for biosynthesis / 中国中药杂志

Sesquiterpene lactones are a kind of widely distributed natural organic compounds with anti-tumor, anti-malarial and other significant biological activities. Based on their carbocylic skeletons, sesquiterpene lactones are classified into germacranolide, guaia-nolide, xanthanolide, pseudo-guaianolide, elemonolide and eudesmanolide, etc. In recent years, with the development of various omics and synthetic biology technologies, the biosynthetic pathways of sesquiterpene lactone compounds of different structural types have gradually been resolved. Among them, the researches on germacrene-derived sesquiterpene lactones are relatively more than others. Therefore, this article focused on the germacrene-derived sesquiterpene lactone biosynthesis pathways and their key enzyme genes, which can lay the foundation for in-depth analysis of sesquiterpene lactone biosynthetic pathways, functional gene mining and heterologous synthesis of active ingredients.

Anti-breast cancer mechanism of transcriptome analysis of ebracteolatain A / 中草药

Objective: To research the mechanism of ebracteolatain A against breast cancer cells, screening the genes with significant changes using second-generation sequencing, and explore the anti-breast cancer mechanism of action of ebracteolatain A at the transcriptomics level. Methods: The acetyl phloroglucinol compound ebracteolatain A was extracted from Euphorbia ebracteolata, interferencing with MCF7 cells (luminal A type of breast cancer cells) to observe differential gene expression between the interfered cells and normal cells. High-throughput transcriptome sequencing and data analysis were performed on three groups of control groups and three experimental groups using Illumina Hi-Seq sequencing technology. Results: A total of 123 656 848, 123 974 262 available reads were obtained in the control group and experimental group, respectively, the reads on the reference genome were 119 762 214, 119 881 622, respectively, accounting for 96.85% and 96.69% of the total; Two groups of transcriptome controls were available: the total number of differential genes was 1 695, of which 770 were up-regulated, 925 were down-regulated, and 3 874 genes were clearly annotated. Bio-enrichment analysis was carried out by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO analysis found that these 3 874 genes mainly involved in biological processes (1 270), cell composition (1 322) and molecular function (1 282), 45 subcategories of three major categories, including cell growth and development, signaling protein activity, membrane and regulation of gene expression. KEGG analysis revealed that the differentially expressed genes involved 263 signaling pathways; The main metabolic pathways were: PI3K-Akt signaling pathway, MAPK signaling pathway, carbohydrate metabolism, myocardial system and cellular reproductive system and etc. Conclusion: The results showed that 1 695 differential genes were screened and identified by Illumina Hi-Seq sequencing technology, and the relationship between the genes of ebracteolatain A and MCF7 cells was further understood, which provided some theoretical cornerstones for breast cancer treatment.

Transcriptomic data analyses of Melicope pteleifolia via Illumina high-throughput sequencing technology / 中草药

Objective: To obtain the transcriptome sequence database of Melicope pteleifolia. Methods: The transcriptome sequencing and systematic bioinformatics analysis were carried out using the second generation high-throughput sequencing platform Illumina HiSeqTM 2000 with mixed root, stem and leaf samples of M. pteleifolia. Results: A total of 47 045 040 high quality sequences (clean reads) were obtained by transcriptome sequencing analysis. A total of 67 956 unigenes were assembled by Trinity de novo, with an average length of 787 nt. BLAST analysis showed that 42 749 (61.92%), 31 152 (45.84%), 26 563 (39.09%), and 17 481 (25.72%) unigenes were annotated in NR, Swiss port, KOG and KEGG databases respectively, and 47 groups were involved in three GO classification: biological process, cellular component and molecular function. A total of 9807 unigenes were annotated to 130 KEGG metabolic pathways, 19 secondary metabolic pathways were screened. Twenty-five different KOG functional groups were obtained by the analysis of KOG functional classification. It was predicted that there were 56 families of higher plant transcription factors. A total of 7 748 simple sequence repeats (SSRs) were found by MISA software. The number (4 117) of the tri-nucleotide SSRs was the richest, with a frequency of 53.1%, and the number of the penta-nucleotide SSRs was relatively small, accounting for 2.2%. Conclusion: The transcriptome information characteristics of root, stem, and leaf of M. pteleifolia can be obtained by high-throughput Illumina sequencing technology and bioinformatics analysis, which will lay a foundation for further research on functional gene mining, secondary metabolic pathway analysis and regulation mechanism of M. pteleifolia.

Molecular genetics research of medicinal plants / 中国中药杂志

With the development of various biotechnology,the research on molecular genetics of medicinal plants has gradually deepened. In this paper,the research system of molecular genetics of medicinal plants was proposed for the first time,which was elaborated from the aspects of genetic resources,genome,gene function and research methods. The application fields of medicinal plant mainly contain species identification,molecular breeding and biosynthesis. The research directions of molecular genetics of medicinal plants in genetic resources,model platform,synthetic biology and molecular breeding were put forward,which include 1 000 genome projects of medicinal plants,model species and mutant libraries,gene original libraries of heterologous synthetic systems,construction gene original library and specific chassis cells in heterologous synthesis system of active ingredient,breeding of new varieties of medicinal plants with high active ingredient and high resistance based on molecular markers andtransgenes.

Research progress on genomics, transcriptomics and functional genes of Dendrobium officinale / 中草药

Dendrobium officinale is one of the most precious medicinal plants in the family of Orchidaceae, and rich in polysaccharides, astragalus, bibenzyls and alkaloids. It has effects such as antioxidant, anti-cancer, immuno-enhancing, lowering blood sugar, and alleviating liver injury. Its huge medicinal, scientific and commercial value has raised a research upsurge, especially in the past five years, nucleic acid molecular biology of Dendrobium officinale has made more and more exciting results. In order to provide scientific guidance for further research on functional genes, this paper reviews the recent progress in research on genomics, transcriptomics and functional genes of D. officinale.

Applications of RNA-Seq technology on medicinal plants / 中草药

RNA-Sequencing (RNA-Seq) is a newly developed method to analyze gene function and interaction at the omics level, which is widely used in frontier field in molecular biology and other fields. In recent years, this technology has been intensively used in the research of medicinal plants, and growing number of reports are published. This paper systematically sorted out relevant literatures and summarized applications of RNA-Seq technology in functional gene discovery, gene network analysis, genetic mechanism revelation and development of molecular marker of medicinal plants. Meanwhile, according to the technical characteristics of RNA-Seq and the development needs of medicinal plant research, this article brings the future prospects regarding RNA-seq technology in Chinese medicinal materials, and intending to provide inspirations for researches on Chinese materia medica based on RNA-Seq.

Transcriptomic data analyses of rhizome of Souliea vaginata via Illumina high-throughput sequencing technology / 中草药

Objective To obtain the transcriptome dataset of rhizome of Souliea vaginata Methods Using the high-throughput illumina sequencing platform Illumina HiSeqTM 2000 150PE, a rhizome transcriptome dataset of S. vaginata was obtained, followed by systemic bioinformatics analyses. Results The transcriptome sequencing analyses produced to a great number of 63 322 086 high quality clean reads. Trinity de novo assembling resulted in a total of 52 575 unigenes with an average length of 909 nt. BLAST analysis indicated that 28 842 (accounting for 54.86% of the total unigenes), 10 712 (20.37%), 9 245 (17.58%), and 11 559 (21.99%) unigenes were successfully annotated in the NR, Swiss-port, KOG, and KEGG databases, respectively. GO classification contained the basic three major groups, including biological process, cellular component, and molecular function, and 45 subgroups. Among them126 KEGG standard pathways were designated, of which 17 were defied as the secondary metabolism. Of all unigenes, 2 215 with protein coding sequences were predicted, and 55 families of plant transcription factors were also identified. MISA prediction yielded a number of 4 609 simple sequence repeats (SSRs), among which the tri-nucleotide SSRs were abundant with 2 106 (45.7%), whereas the penta-nucleotide SSRs were relatively less, accounting for 2.9%. Conclusion The transcriptomic characteristics of S.vaginata rhizome were revealed by the high-throughput Illumina sequencing technology along with bioinformatics analyses, which would be of great importance for the functional gene characterization, secondary metabolism pathway dissections, and their regulatory mechanisms in S. vaginata.

Transcriptomic analyses of Chloranthus japonicus by RNA-seq / 中草药

Objective To obtain the transcriptome dataset of Chloranthus japonicas. Methods Using the Illumina HiSeqTM 2000 150PE, a rhizome transcriptome of C. japonicus was generated, followed by systemic bioinformatics analyses. Results A total of 68 458 750 high quality clean reads were produced by the transcriptome sequencing. Trinity de novo assembling resulted in a total of 56 096 unigenes with an average length of 801 nt. BLAST analysis indicated that 25 773 (45.94%), 17 801 (31.73%), 16 082 (28.67%), and 9 649 (17.20%) unigenes were successfully annotated in the NR, Swiss-port, KOG, and KEGG databases, respectively. All unigenes were classified into three major groups by GO, including biological process, cellular component, and molecular function, and then, grouped into 40 subgroups. And 131 KEGG standard pathways were designated, 16 of which were defied as the secondary metabolism. Further analysis revealed that a total of 170 unigenes were involved in the biosynthesis of mono-, di-, sesqui-, or triterpene. Meantime, 1 887 unigenes were predicted to contain protein coding sequences. Totally 54 families of transcription factors of higher plant were identified. Using MISA prediction, 8 987 simple sequence repeats (SSRs) were obtained, among which the di-nucleotide SSRs were abundant with 5 948 (66.2%), whereas the penta-nucleotide SSRs were relatively less, accounting for 1.3%. Conclusion The transcriptome of C. japonicus rhizome was generated by RNA-seq along with the identification of unigenes implicated in various terpenes biosynthesis, which will provide a fundamental basis for secondary metabolism pathway dissections and their regulatory mechanisms in this plant species.

“Preventing transform when sickness”——Regulating pre-metastasis niche to prevent tumor metastasis / 中草药

Metastasis is the leading cause of cancer-related death. Pre-metastatic niche formation is the contributing factor of tumor metastasis. Traditional Chinese medicine (TCM) has a curative effect on metastasis and cancer recurrence in clinic. Modern pharmacological studies have shown that China meteria medica (CMM) can inhibit tumor metastasis by affecting tumor secretion, preventing recruitment of immune suppressive cells, and influencing anti-inflammatory polarization of matrix components in certain tissues. The regulation of CMM has the characteristics of multi-target, minor effect, and bidirection, It may play a integrate role with multi-factor and minor effect in regulating tumor-related gene expression or gene-gene combination by influencing regulating tumor-related functional gene networks. This is consistent with the research strategy of taking signal transduction dynamic network as drug target. Therefore, the prevention and treatment of pre-metastatic niche formation via TCM is an effective research strategy. This review summarizes the current research progress on regulating pre-metastasis niche by CMM, and provides a theoretical basis for the future research of TCM to prevent tumor metastasis.

Sequential analysis of genome of Thelazia callipaeda / 中国血吸虫病防治杂志

Objective To investigate the molecular characteristics of genome sequence of Thelazia callipaeda(T.cp).Meth-ods The obtained T.cp genome assembling data were annotated by using a combination of ab initio gene by softwares,Gene-Mark and GeneID,and the homology of the experimentally confirmed genes was predicted by software GeMoMa.The results were integrated by software EVM to predict all genes of genome.The obtained genes were annotated in the common public data-base and three dedicated databases(CAZyme,TCDB and PHI),respectively.Results The Scaffolds and Contigs gene struc-ture of T.cp genome(79.34 Mb)was analyzed,and a total of 6 333 genes were obtained.The sequence search was conducted in the public databases using BLASTx,of which 97.85％of the genes could be annotated.The genes annotated in the NR database were the most(98.69％),and those enriched in the KEGG pathway were the least(50.50％).The functional genes were blasted by KOG database and totally 4 517 genes were found.The three special databases(CAZyme,TCDB and PHI)were used to an-notate all the genes,and 136,139 and 1 498 genes were assigned respectively,and the number of genes in the PHI database was the largest.In the cytochrome proprietary database,238 cytochrome P450 genes were predicted.Conclusion We have pre-liminarily revealed the T.cp genome structure characteristics and annotation information,and totally 6 333 genes are obtained.

Production of Active Compounds in Medicinal Plants: From Plant Tissue Culture to Biosynthesis / 中草药·英文版

Over past decades plant tissue culture has emerged as an alternative of whole plant cultivation in the production of valuable secondary metabolites. Adventitious roots culture of Panax ginseng and Echinacea purpure has reached the scale of 1-10 kL. Some molecular biological techniques, such as transgenic technology and genetic stability are increasingly used in the studies on plant tissue cultures. The studies on elicitors have deepened into the induction mechanism, including signal molecules, functional genes, and so on. More and more biological elicitors, such as A. niger and yeast are used to increase the active compounds in plant tissue cultures. We also discussed the application of synthetic biology in the studies on biosynthesis of artemisinin, paclitaxel, and tanshinon. The studies on active ingredients biosynthesis of medicinal plants provide unprecedented possibilities to achieve mass production of active ingredients. Plant tissue cultures can not only produce active ingredients but also as experimental materials for biosynthesis. In order to improve the contents of active compounds in medicinal plants, following aspects could be carried out gene interference or gene silencing, gene overexpression, combination with chemical synthesis, application of elicitors, and site-directed mutagenesis of the key enzymes.

Study on the cDNA Library Construction Method for Xinjiang Artemisia rupestris / 中国药房

OBJECTIVE:To establish a method for full-length cDNA library of Xinjiang Artemisia rupestris. METHODS:Mod-ified Trizol method was adopted to extract total RNA in young leaves of A. rupestris,it was transcribed into single-strand cDNA, and then synthesized into double-strand cDNA by long-distance polymerase chain reaction(LD-PCR)method. PCR product was di-gested by proteinase K and sfiⅠ,and then fractionated by CHROMA SPIN-400 columns. The cDNA longer than 0.4 kb were col-lected and ligated to phage λTriplE × 2,and then protein packaging was performed. Full-length cDNA library was established by SMART technology. 20 monoclonal were randomly selected from the library,and electrophoresis was used to determine the primary library titer,library capacity,recombinant positive rate and length of insert cDNA. RESULTS:The primary library titer was 1.94× 107 pfu/mL,library capacity was 0.97×107 pfu;recombinant positive rate of insert cDNA was 96% and length was 0.5-2.0 kb with an average of 0.9 kb. CONCLUSIONS:The established library is high in capacity and quality,which can provide basis for estab-lishing cDNA library of Xinjiang A. rupestris.

Advance in molecular biology of Dendrobium (Orchidaceae) / 中国中药杂志

With the development of molecular biology, the process in molecular biology research of Dendrobium is going fast. Not only did it provide new ways to identify Dendrobium quickly, reveal the genetic diversity and relationship of Dendrobium, but also lay the vital foundation for explaining the mechanism of Dendrobium growth and metabolism. The present paper reviews the recent process in molecular biology research of Dendrobium from three aspects, including molecular identification, genetic diversity and functional genes. And this review will facilitate the development of this research area and Dendrobium.

Data mining and influential analysis of gene expression data for plant resistance gene identification in tomato (Solanum lycopersicum)

Background Molecular mechanisms of plant-pathogen interactions have been studied thoroughly but much about them is still unknown. A better understanding of these mechanisms and the detection of new resistance genes can improve crop production and food supply. Extracting this knowledge from available genomic data is a challenging task. Results Here, we evaluate the usefulness of clustering, data-mining and regression to identify potential new resistance genes. Three types of analyses were conducted separately over two conditions, tomatoes inoculated with Phytophthora infestans and not inoculated tomatoes. Predictions for 10 new resistance genes obtained by all applied methods were selected as being the most reliable and are therefore reported as potential resistance genes. Conclusion Application of different statistical analyses to detect potential resistance genes reliably has shown to conduct interesting results that improve knowledge on molecular mechanisms of plant resistance to pathogens.

"Reverse Immunology" and The Discovery of Immune-related Functional Genes / 生物化学与生物物理进展

The completion of sequencing human genome creates a new era of biological science and technology. Although the sequence of the human genome has been known, it is still hard to rapidly explore the whole functional genes, especially, their interaction with each other and the meaning to the body. However, the "reverse biology" which comes into being in the recent years provides us a series of novel ideas and technologies for discovering new functional gene, among which the immune-related genes have attracted more attentions, clarifying how functional gene works and their potential value in application.

The Application of Genosensor in the Detection of Functional Genes of Environmental Microorganisms / 微生物学通报

The fast development of the molecular biology and the further research on the nucleic acid set a solid foundation for the development of genosensor.Genosensor is the result of combining molecular biology with microelectronics,electrochemistry,optics and etc,which will build a bridge between the life science and the information science and become one of the most important technologies for DNA information analysis and detection.The working principle,classification of genosenor and the recent research on its application in the detection of functional genes of environmental microorganisms are discussed according to the latest literature.And it is pointed out that the application in the determination of microorganism functional genes in compost is an important development orientation of genosensor.