Construction of aseptic cultural system of Salvia miltiorrhiza and determination of active components by HPLC analysis / 中草药

Objective: The regeneration system of Salvia miltiorrhiza was constructed and contents of active components of the sterile materials were determined, so as to provide the basis for the rapid propagation and secondary metabolism regulation of S. miltiorrhiza. Methods: The optimum hormone ratio for inducing callus was screened by orthogonal test. The buds and roots induction were conducted to estabilish the tissue culture system. The contents of effective components were evaluated by HPLC analysis. Results: The suitable medium for callus induction was MS+6-BA (2.0 mg/L)+NAA (1.0 mg/L)+2,4-D (0.5 mg/L). The preferred enrichment medium of adventitious bud induction was MS+6-BA (2.0 mg/L)+NAA (1.0 mg/L). And rooting medium was 1/2 MS+NAA (0.5 mg/L). Seven active components in aseptic seedlings, callus, and regenerated seedlings could be detected with significant differences in different aseptic materials (P < 0.05). The contents of rosmarinic acid and salvianolic acid B were higher than the others. Conclusion The culture system of S. miltiorrhiza was successfully established, and vigor regenerated seedlings were also obtained. The accumulation of active components in the three sterile materials showed difference, laying a foundation for further study in S. miltiorrhiza.

Investigation on seeds characteristics and establishment of aseptic cultural system for Rheum palmatum / 中草药

Objective: To study the seeds shapes and germination characteristics, construct aseptic seedlings and callus cultural system, and provide a basis for the rapid propagation and secondary metabolic regulation of Rheum palmatum. Methods: Ten batches of R. palmatum seeds were subjected to characteristic analyses from the aspects of size, purification, weights per thousand seeds, seeds vigor, germination rates, and germination energy. The optimum disinfection system for aseptic seedlings and the optimum hormone ratio for inducing callus were screened by orthogonal test. The content of ten active components in aseptic seedlings and calli were primarily evaluated by HPLC analysis. Results: There was no significant difference in the appearances of the ten batches of R. palmatum seeds from different regions. The content of moisture, seeds vigor, germination rates, and germination energy differed apparently. The germination characteristics in Hezheng and Weiyuan counties were the best. The best disinfection group for aseptic seedlings was the combination of 75% ethanol for 30 s with 10% hydrogen peroxide for 15 min. The optimum hormones for callus induction were 6-BA (1.0 mg/L) + KT (2.0 mg/L) + NAA (1.5 mg/L). Seeds treated with different disinfectants had no significant effect on the content of ten components in germinated aseptic seedlings (P > 0.05). Seven active components were detected in callus, which was significantly lower than that in aseptic seedlings. And the content of chrysophanol-8-O-glucoside was the highest in the callus. Conclusion: The seed characteristics from Hezheng and Weiyuan counties in Gansu Province were excellent by analyses of weights per thousand seeds, seeds vigor, germination rates, and germination energy. The aseptic seedlings and callus cultural system of R. palmatum were successfully established, laying a solid foundation for further study.

High-throughput transcriptomic sequencing of Rheum palmatum L. seedlings and elucidation of genes in anthraquinone biosynthesis / 药学学报

Anthraquinones are not only the main active constituents but also the index components for the quality control of Rhei Radix et Rhizoma. To study the anthraquinone biosynthesis, Rheum palmatum L. seedlings were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM 2000 150PE. The Illumina sequencing generated a total of 11.04 G clean data resulting in 736 309 74 clean reads, deposited in the sequence read archive (SRA accession SRP160030). Trinity do novo assembly yielded 93 646 unigenes, with an average of 1 108 nt. Functional annotation revealed that all unigenes were successfully annotated in the NR, NT, Swiss-port, PFAM, and KOG databases. GO enrichments showed that 57 subgroups were involved in biological process, cellular component, and molecular function. KEGG analysis indicated that 1 107 unigenes were implicated in 19 standard secondary metabolic pathways. 172 unigenes were analyzed to encode 28 key enzymes during the MVA, MEP, shikimic acid, and polyketide pathways related to anthraquinone biosynthesis. 125 CYP450 and 73 UGTs unigenes were related the modification of secondary metabolites in R. palmatum L. Furthermore, seven unigenes with full length cDNAs were successfully verified by RT-PCR and sequencing analyses. Then, MISA prediction produced a number of 18 885 simple sequence repeats (SSRs). Herein, the transcriptomic gene expression profiles of R. palmatum L. and candidate genes during the anthraquinone biosynthesis pathway were obtained for the first time. The results provided basic information for subsequent gene function characterization, secondary metabolic pathway analysis, and anthraquinone biosynthesis and regulation elucidation in R. palmatum L.

Identification and expression analysis of four CIPKs genes from a rare and medicinal plant, Dendrobium officinale / 药学学报

The calcineurin B-like protein (CBL)-interacting protein kinase (CIPK) plays a vital role in the growth, development, and stresses adaptation in plants by interaction with the calcium signaling. In this study, four full length cDNAs of CIPKs genes, namely DoCIPK1, DoCIPK2, DoCIPK3 and DoCIPK4 (GenBank accession No. KT957557, KT957558, KT957559 and KT957560, respectively) were cloned from the rear and medicinal plant, Dendrobium officinale, by rapid amplification of cDNA ends (RACE) for the first time. The corresponding encoded proteins, consisting of 473, 449, 451 and 440 amino acids (aa), respectively, with a molecular weight of 53.50, 50.93, 51.50 and 50.16 kDa, and an isoelectric point (pI) of 7.99, 9.25, 8.81 and 9.11, respectively, shared 70%-90%, 69%-80%, 78%-93%, and 66%-82% identities CIPKs with various plants. Each deduced protein contained a conserved protein kinase domain (respectively at 21 -275, 14-268, 16-271 and 12-266 aa position), a CIPKs family characteristic NAF/FISL domain (respectively at 335-391, 313-370, 310-369 and 305-362 aa position) and some functional motifs. The four DoCIPK proteins, without signal peptide or transmembrane region, were located in the plasma membrane and endoplasmic reticulum at the subcellular level. The three dimensional structure of the proteins were similar to that of Arabidopsis AtCIPK24. DoCIPK1 and DoCIPK3 were respectively clustered in the group E and A of the Arabidopsis and rice CIPK evolutionary tree, while DoCIPK2 and DoCIPK4 belonged to group C. The relative expression of DoCIPK1 showed no significant difference in the leaves and stems, and its transcripts in the roots was 0.35 fold over that in the leaves. The abundance of DoCIPK3 transcripts in the stems and the roots were 3.36 fold and 3.47 fold higher, respectively, than those in the leaves. DoCIPK2 exhibited similar expression pattern to DoCIPK4. Their relative expression in the leaves and the stems had no apparent difference, and the transcript levels were higher in the roots than that in the leaves, with 2.08 fold and 7.86 fold, respectively. Cloning, bioinformatics analyses, and expression patterns of the four DoCIPK genes provide a basis for functional elucidation of these genes further during the physiological responses in D. officinale.

Isolation and expression analysis of a hexose transporter gene DoHT1 in Dendrobium officinale / 中国中药杂志

In this study, RACE technology was employed to isolate the full length cDNA of DoHT1 in Dendrobium officinale, followed by bioinformatics analysis of the sequence characteristics. And the expression pattern of the gene was also analyzed by quantitative PCR. The full length cDNA of DoHT1 was 1 586 bp in length, containing a 1 536 bp ORF, which encoded a 511-aa protein with molecular weight of 56.18 kD and isoelectric point of 9.08. The deduced DoHT1 protein had the major facilitator superfamily conserved domain (22-483), SUGAR₋TRANSPORT₋1 (139-164), and SUGAR₋TRANSPORT₋2 (338-355), typical for sugar transporter; DoHT1, without a single peptide had 11 transmembrane regions, and was predicted to locate in the plasma membrane; DoHT1 had high identities (54.7%-80.7%) with HTs proteins from various plants. DoHT1 belonged to the MST (monosaccharide transporter) group of the evolutionary tree, and was closely related to the Phalaenopsis equestris. DoHT1 was differentially expressed in the three included organs. The transcripts were significantly the most abundant in the leaves with 19.36 fold than roots, then 1.82 fold in the stems than the roots. The identification and molecular characterization of the full length DoHT1 will be essential for further function study of the gene during the regulation of sugar metabolism of D. officinale.