Screening of reference genes for quantitative real-time PCR in Artemisia argyi / 中国中药杂志

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.

Genome-wide Identification and Characterization of TIFY Gene Family in Medicinal Plant Cannabis sativa / 中国实验方剂学杂志

Objective:The TIFY gene family will be identified and characterized from the whole genome level in Cannabis sativa，which will lay the foundation for gene function study on TIFY family genes and their regulation mechanism on the biosynthesis of cannabinoids and other secondary metabolites. Method:Using the existing genomic data of cannabis，the CsTIFY genes were identified through bioinformatics analysis tools such as NCBI，PlantTFDB，MEME and TBtools etc.，and physicochemical properties，phylogenetic trees，gene structures，chromosome locations and gene expression patterns were analyzed and visualized. Result:Fourteen TIFY family genes（CsTIFY1-CsTIFY14） were identified in Cannabis sativa，which belong to four subfamilies：TIFY，JAZ，ZML，and PPD. The CsTIFYs are composed of 365-1 369 bp nucleotides encoding 118-442 amino acid residues，and their isoelectric points are 4.64-9.96. The 14 CsTIFYs are unevenly distributed on 8 chromosomes，and their proteins are all located in the nucleus. The promoter of CsTIFYs contain multiple abiotic stress responsive cis-acting elements，which indicated that CsTIFYs might involved in the regulation of different abiotic stresses. Transcriptome profiling revealed that CsTIFYs expressed differently in female flowers of 10 differently cannabis varieties，or in flowers，bracts，stems，and leaves of the same variety. Conclusion:Fourteen TIFY family genes were characterized from the whole genome level in C. sativa，and their phylogenetic evolutions and gene expression patterns were analyzed，indicating that CsTIFYs may play important regulatory roles in JA signal transduction，abiotic stress and cannabinoid biosynthesis. This study will provide valuable reference for gene function study of the TIFY family genes in cannabis and cannabis breeding.

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.

Identification of 23 unknown Li minority medicinal plants based on DNA barcoding / 中国中药杂志

DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.

Application of DNA metabarcoding in species identification of Chinese herbal medicines / 中国中药杂志

DNA metabarcoding,one rapid and robust method using specific standard DNA fragments,has been widely used for rapid species identification of a bulk sample through high-throughput sequencing technologies.While it has been widely used in the studies of metagenomics,animal and plant biodiversity,it has gradually come to be used as a profitable method in species identification of mixed Chinese herbal medicines.In this paper,we mainly summarize the current studies of the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines.Moreover,high-throughput sequencing technologies adopted in those studies,such as Sanger,the next-generation,and third-generation sequencing technologies,are discussed.It is conducted to provide a theoretical guidance for the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines and in more other biodiversity studies.

Influence of light on gene expression of key synthesis enzyme genes FtANR and FtLAR about proanthocyanidin in seeds of homologous plant of food and medicine Fagopyrum tataricum / 中国中药杂志

Tartary buckwheat Fagopyrum tataricum is an important medicinal and functional herb due to its rich content of flavonoids in the seeds. F.tataricum exhibited good functions for free radicals scavenging， anti-oxidation， anti-aging activities. Although much genetic knowledge of the synthesis， regulation， accumulation of rutin， the genetic basis of proanthocyanidins(PAs) in tartary buckwheat and their related gene expression changes under different lights(blue， red， far red， ultraviolet light) remain largely unexplored. In this study， we cloned one anthocyanidin reductase gene(ANR) and two leucocyanidin reductase gene(LAR) named FtANR，FtLAR1，FtLAR3 involved in formation of(+)-catechin and(-)-epicatechin precusor proanthocyanidin by digging out F. tataricum seed transcriptome data. The expression data showed that the opposite influence of red light on these gene transcript level compared to others lights. The expression levels of FtANR and FtLAR1 decreased and FtLAR3 appeared increment after exposed in the red light， while the expression levels of those genes appeared opposite result after exposed in the blue and far red light.

Identification of herbal tea ingredient Mesona chinensis and its adulterants using ITS2 barcode / 中国药学杂志

OBJECTIVE: To establish an accurate and rapid method for identifying Mesona chinensis and its adulterants using the internal transcribed spacer 2 (ITS2) region as a barcode. METHODS: The total genomic DNA from 55 samples representing five species and four variants including M. chinensis and its adulterants were extracted. The ITS2 regions were amplified, and the complete ITS2 sequences were annotated by CodonCode Aligner. The intra- and inter-specific genetic distances based on Kimura 2-Parameter (K2P) and the Neighbor-Joining (NJ) phylogenetic tree were analyzed to identify the species. RESULTS: The lengths of all the 1TS2 sequences of M. chinensis were 232 bp. The intra-specific genetic distances (0) were far shorter than the inter-specific ones between M. chinensis and its adulterants (0.210 2-0.301 9). NJ tree analysis indicated that M. chinensis were accurately differentiated from its adulterants. CONCLUSION: The ITS2 region as an DNA barcode can accurately and effectively distinguish herbal tea ingredient M. chinensis from its adulterants, which provides a quick identifying method to ensure its safe use.

Identification of herbal tea ingredient Plumeria rubra and its adulterants using DNA barcoding / 中国中药杂志

ITS2 sequence was used as a barcode to identify herbal tea ingredient Plumeria rubra and its adulterants. Genomic DNAs from forty eight samples were extracted, the ITS2 sequences were amplified and sequenced bi-direstionlly, and then assembled and obtained using CodonCode Aligner. The sequences were aligned using ClustalW, the genetic distances were computed by kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic trees were constructed using MEGA5.0. Results showed that the length of ITS2 sequence of P. rubra were 244 bp. The intra-specific genetic distances (0-0. 016 6) were much smaller than inter-specific ones between P. rubra and its adulterants(0.320 8-0.650 4). The NJ tree indicated that P. rubra and its adulterants could be distinguished clearly. Therefore, Using ITS2 barcode can accurately andeffectively distinguish herbal tea ingredient P. rubra from its adulterants, which providesa new molecular method to identify P. rubra and ensure its safety in use.