Comparative analysis in different organs and tissue-specific metabolite profiling of Atractylodes lancea from four regions by GC-MS and laser microdissection.

Traditional Chinese medicine is made from the rhizome of Atractylodes lancea (Thunb.) DC. (Compositae), known as Cangzhu. In this study, gas chromatography-mass spectrometry was used to identify and quantify the volatile oils of different organs of A. lancea from four regions of China: Jiangsu, Anhui, Henan, and Hubei provinces. The volatile oils of A. lancea were qualitatively and quantitatively characterized using gas chromatography-mass spectrometry combined with laser microdissection. The results identified 21 components in A. lancea, the majority of the components were found in the rhizomes, followed by the fibrous roots, flowers, leaves, and stems. According to the contents of volatile oils in A. lancea, it was divided into Dabieshan (mainly includes hinesol and ß-eudesmol) and Maoshan types (mainly includes atractylon and atractylodin), and the ratios of hinesol:ß-eudesmol:atractylon:atractylodin were 17.06:4.55:0:1, 12.66:11.71:0.99:1, 7.43:6.23:0:1, and 0.13:0.16:1.52:1 in A. lancea from AH, HN, HB, and JS, respectively. Tissue-specific study indicated that Dabieshan type mainly includes elemol, hinesol, and ß-eudesmol in the periderm and secretory cavities of A. lancea, whereas Maoshan type mainly includes atractylon, atractylodin, little hinesol, and ß-eudesmol in the secretory cavities. Conversely, no volatile oils were detected in the cortex, phloem, xylem, vascular ray, or pith. This study provides a foundation for further evaluation and utilization of A. lancea.

Genome survey sequencing of Atractylodes lancea and identification of its SSR markers.

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine rich in sesquiterpenes that has been widely used in China and Japan for the treatment of viral infections. Despite its important pharmacological value, genomic information regarding A. lancea is currently unavailable. In the present study, the whole genome sequence of A. lancea was obtained using an Illumina sequencing platform. The results revealed an estimated genome size for A. lancea of 4,159.24 Mb, with 2.28% heterozygosity, and a repeat rate of 89.2%, all of which indicate a highly heterozygous genome. Based on the genomic data of A. lancea, 27,582 simple sequence repeat (SSR) markers were identified. The differences in representation among nucleotide repeat types were large, e.g., the mononucleotide repeat type was the most abundant (54.74%) while the pentanucleotide repeats were the least abundant (0.10%), and sequence motifs GA/TC (31.17%) and TTC/GAA (7.23%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. A total of 93,434 genes matched known genes in common databases including 48,493 genes in the Gene Ontology (GO) database and 34,929 genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This is the first report to sequence and characterize the whole genome of A. lancea and will provide a theoretical basis and reference for further genome-wide deep sequencing and SSR molecular marker development of A. lancea.

[Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida].

In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.

[Identification of Corydalis yanhusuo,C. turtschaninovii,C. decumbens by allele-specific PCR].

To establish a DNA molecular markers method for identification of Corydalis yanhusuo,C. turtschaninovii and C. decumbens,the mat K,trn G and psb A-trn H sequences of 56 samples from 14 species of C. yanhusuo,C. turtschaninovii,C. decumbens and their related species were obtained by sequencing. The SNP loci were obtained by Bio Edit 7. 2. 2 software. The primers for AS-PCR identification were designed based on the mutation sites,and the conditions of PCR were optimized to identify C. yanhusuo,C. turtschaninovii,and C. decumbens according to the specific bands. The results showed that the amount of template( 0. 6-1 200 ng)and annealing temperature( 42-60 ℃) had little influence on the amplification results,and the number of cycles had much influence on the amplification results. When the number of cycles was 20,the specific bands of 297 bp( mat K),353 bp( trn G) and 544 bp( mat K) were amplified from C. yanhusuo,C. turtschaninovii and C. decumbens,respectively. The method established in this study had a minimum detection limit of 6 ng for C. yanhusuo,60 ng for C. decumbens and less than 0. 6 ng for C. turtschaninovii. Thus,the allelespecific PCR method established in the research can specifically identify C. yanhusuo,C. turtschaninovii,and C. decumbens.

[Species and medical history of "Xishuang" medicinal materials].

"Xishuang" is a special phenomenon that chemical composition of medicinal materials crystallize on the surface exposed to air for a long time. We summarized Herbal textual research of "Xishuang" phenomenon of six herbs, such as Schisandrae Chinensis Fructus, Moutan Cortex, Atractylodis Rhizoma, Magnoliae Officinalis Cortex, dried persimmon frost and watermelon frost. From historical perspective, cream of Schisandrae Chinensis Fructus was firstly discovered in Lei Gong's Moxibustion Theory. Thereafter, dried persimmon frost was found in Song Dynasty, which was named "white persimmon" in Ben Cao Tu Jing and had become an independent medicine in Compendium of Materia Medica. Then, watermelon frost was found in Yang Yi Da Quan of the Qing Dynasty, and Moutan Cortex's "sand star" was recorded in Zeng Ding Wei Yao Tiao Bian of the Republic of China. After that, "Xishuang" phenomenon of Atractylodis Rhizomaand Magnoliae Officinalis Cortex were reported in 1950s and 1960s in succession. The pattern of "Xishuang" is divided into different type, natural "Xishuang" includes Schisandrae Chinensis Fructus, Moutan Cortex, Atractylodis Rhizoma and Magnoliae Officinalis Cortex, artificial "Xishuang" includes watermelon frost, and dried persimmon frost formed crystals by using artificial intervention. The above 6 kinds of herbs have different crystal structure and chemical composition. Therefore, according to traditional identification experience, "Xishuang" phenomenon is related to varieties and quality of medicinal herbs. These research provide herbalism basis for the modern study of "Xishuang" medicinal materials.