This study was aimed to screen candidate genes involved in the triterpenoid saponins biosynthetic pathway of the Ilex asprella root. The Illumina platform was applied to perform transcriptomic sequencing of I. asprella root, followed by a series of bioinformatics analysis. The results showed that a total of 272 candidate unigenes were anno-tated to be involved in the biosynthetic pathway of terpenoid in the transcriptome of I. asprella root, including 72 u-nigenes for the upstream pathway and 26 unigenes for cyclization, oxidation and glycosylation in the downstream pathway. Phylogenetic analysis was carried out to further analyze the evolution relationship of some candidate uni-genes and their homologous genes. Two genes IaA S1 and IaA S2 were proved to be mixed amyrin synthases in yeast expression system. Moreover, IaA S1 was identified to one of the rare ASs with α-amyrin as the major product. It was concluded that a series of candidate genes, which might be involved in the biosynthetic pathway of triterpenoid saponins, were screened out from the transcriptome of I. asprella root. Further investigation of these candidate genes will provide insight into their actual functions in the triterpenoid saponins biosynthetic pathway in I. asprella.

Ultrafine powder and cell wall-broken powder of herbal medicine lack of the morphological characters and microscopic identification features. This makes it hard to identify herb's authenticity with traditional methods. We tested ITS2 sequence as DNA barcode in identification of herbal medicine in ultrafine powder and cell wall-broken powder in this study. We extracted genomic DNAs of 93 samples of 31 representative herbal medicines (28 species), which include whole plant, roots and bulbs, stems, leaves, flowers, fruits and seeds. The ITS2 sequences were amplified and sequenced bidirectionally. The ITS2 sequences were identified using Basic Local Alignment Search Tool (BLAST) method in the GenBank database and DNA barcoding system to identify the herbal medicine. The genetic distance was analyzed using the Kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0. The results showed that DNA can be extracted successfully from 93 samples and high quality ITS2 sequences can be amplified. All 31 herbal medicines can get correct identification via BLAST method. The ITS2 sequences of raw material medicines, ultrafine powder and cell wall-broken powder have same sequence in 26 herbal medicines, while the ITS2 sequences in other 5 herbal medicines exhibited variation. The maximum intraspecific genetic-distances of each species were all less than the minimum interspecific genetic distances. ITS2 sequences of each species are all converged to their standard DNA barcodes using NJ method. Therefore, using ITS2 barcode can accurately and effectively distinguish ultrafine powder and cell wall-broken powder of herbal medicine. It provides a new molecular method to identify ultrafine powder and cell wall-broken powder of herbal medicine in the quality control and market supervision.

Objective This study aimed to investigate the glycosyltransferase gene DsUGT11 involved in the biosynthesis of flavonoids in Desmodium styracifolia,and to analyze the function of its encoding protein by bioinformatic tools,gene cloning,prokaryotic expression,and other technologies.Methods The sequence characteristics and potential biological functions of DsUGT11 were analyzed and predicted by bioinformatics analysis,respectively.Total RNA was extracted from fresh leaves and reverse transcribed into cDNA,from which DsUGT11 gene was successfully amplified and cloned.Heterologous expressed protein was induced and purified,followed by functional characterization using enzymatic reaction in vitro.Results A candidate glycosyltransferase gene,designated as DsUGT11,was identified from the transcriptome data of D.styracifolia.The length of the open reading frame of DsUGT11 is 1426 bp,and the molecular weight of its encoding protein is expected to be 52.14 KDa.By bioinformatic analysis,DsUGT11 was found to harvest a conserved motif of"PSPG"that is unique to the UGT family.Moreover,DsUGT11 was successfully amplified and cloned using the prokaryotic expression vector pMALc5X.Recombinant protein was induced and purified subsequently.Next,the purified protein was used to perform the enzymatic reaction in vitro,the result of which suggested that DsUGT11 was able to catalyze the conversion of 2-OH-naringenin and UDP-glucose into three different compounds,one of which was authenticated as apigenin-7-O-β-D-glucoside(also known as Apigetrin),with two others unknown.Conclusion In this study,the DsUGT11 gene was identified and cloned,whose encoding protein is a flavone-oxyglycosyltransferase catalyzing the conversion of 2-OH-naringenin and UDP-glucose into three different compounds including Apigetrin.