Estimation of genome sizes of Astragalus membranaceus based on flow cytometry and K-mer analysis / 中草药

Objective: To determinate the genome size and complexity of Astragalus membranaceus by using flow cytometry (FCM) and K-mer analysis, which can lay the foundation for the screening of functional genes of A. membranaceus. Methods Lycopersicon esculentum was served as an internal reference in this study. The mixed sample of A. membranaceus cell nucleus and L. esculentum cell nucleus was stained using propidium iodide (PI). The PI fluorescence intensities of the sample were measured by FCM. The genome size of A. membranaceus was calculated by comparing the multiple relationship between the peak of DNA content in the cells of A. membranaceus and L. esculentum. The genome of A. membranaceus was sequenced by using high-throughput sequencing technologies. The genome size of A. membranaceus was calculated by K-mer analysis. The hybridity percentage, repetitive sequence, and GC of A. membranaceus were estimated by bioinformatics analysis. Results The genome size of A. membranaceus was about 1 426 Mb. For K-mer analysis, more than 95 Gb high quality data from the genome was generated. The average genome size and sequencing coverage depth of A. membranaceus was about 1 456 Mb and 39 times respectively. The genome of A. membranaceus had obvious hybridity peak by K-mer method, and the hybridity percentage as high as 2.1%. Conclusion The genome size of A. membranaceus was about 1.45 Gb and the heterozygosity is high. These data would provide a reference for the genomic research in A. membranaceus.

Optimization of extraction process of Shenqi Qiangxin Tablets based on pharmacodynamic test and analytic hierarchy process / 中草药

Objective: To optimize the extraction technology of Shenqi Qiangxin Tablets (SQT). Methods: With the improvement of heart lesion of pharmacological model of isoproterenol induced heart failure in rats as the index, pharmacological efficacy test was used to screen extracting conditions of the technology. The extraction technology was optimized by analytic hierarchy process combined with principal component analysis, single factor and orthogonal tests using each content of solid matter, ginsenosides Rg1, Re as indexes. And the verification test was carried out by using solid mass and icariin content as indexes. Results: Pharmacological efficacy test showed that technology 4 was superior. The optimal extraction condition of technology 4 was as follow: five medicinal materials including red ginseng and astragalus were reflux extracted three times with 50% ethanol, 11 fold for the first time, 10 fold for the second and three times, 2.5 h for each extraction; Epimedium and the other two medicinal materials were decocted three times with water, 19 fold for the first time, 16 fold for the second and third times, 1.5 h for each decction. The verification test showed that the average yield of ethanol extracted solids was 19.78%, and the average extraction rate of ginsenoside Rg1 and Re was 77.52%; The average value of water extracted solids was 16.58%, and the average extraction rate of epimedium was 90.98% (RSD < 2.0%, n = 3). Conclusion: The optimized extraction technology was stable and feasible.

Prediction of microRNAs and drought expression pattern analysis in Astragalus membranaceus / 中草药

Objective: To provide a good start for the study of miRNA in Astragalus membranaceus, the miRNAs and their targets were predicted using bioinformatics approach, then the drought-induced expression pattern was analyzed using quantitative RT-PCR. Methods: The deep sequencing data downloaded from public database were assembled using Trinity to establish the transcriptome database. Bioinformatics method was employed to predict miRNAs and their targets, functional classification analyses of targets were conducted. Stem-loop fluorescence quantitative PCR method was used to validate miRNAs, and the drought-induced expression patterns of miRNA were also determined. Results: A transcriptome database with 88 263 sequences was obtained. Based on the transcriptomic sequences, 17 miRNA, classified into 17 families, generated from 17 stem-loop precursors, were identified. A total of 145 genes were predicted to be regulated by these miRNAs, and these genes were involved into diversified biological processes including gene transcription regulation, substance metabolism, signal transduction, stress response, and post translational modification of protein. Eight miRNAs were randomly selected for expression analysis and the results indicated that miR2118 and miR166 might be involved in drought stress response. Conclusion: The miRNAs, the corresponding targets and the drought response miRNAs identified in this study will provide a solid basis for understanding the biological functions of miRNAs in A. membranaceus.

Analysis on codon bias of farnesyl pyrophosphate synthase gene in Astragalus membranaceus / 中草药

Objective: Farnesyl pyrophosphate synthase (FPS) is the key enzyme in biosynthesis pathway of astragaloside IV. The purpose of the experiment was to provide the theory basis for selecting appropriate expression systems and regulating the content of astragaloside IV. Method: FPS gene coding sequence was cloned based on Astragalus membranaceus from Changbai Mountain. Synonymous codons usage of FPS gene was analyzed by EMBOSS and Codon W programs and compared with the genome of other seven plants, such as Zea mays and Artemisia apiacea, and E. coli. Results: FPS gene of A. membranaceus was bias toward the codon with A and T at the third codon position and there are 22 codons showing the significant differences between FPS gene of A. membranaceus and E. coli genome. Conclution: The codons need to be optimized to improve the expression level of FPS gene in E. coli.

Isolation and purification of a lectin from roots of Astragalus membranaceus / 中草药

Objective To isolate and purify a lectin from the roots of Astragalus membranaceus.Methods The protein was purified using a combination of 20%—60% ammonium sulfate fraction and ConA-Sepharose 4B affinity chromatography.Results The purified protein appeared as a single band with molecular mass of 3.15?104 on SDS-PAGE and the relative molecular mass was estimated by gel filtration on a calibrated Superdex 75 column with apparent molecular weight of 3.35?104.This lectin was a glycoprotein with a neutral carbohydrate content of 10.7%.Conclusion A lectin is isolated and purified from the roots of A.membranaceus for the first time.It is a monomer glycoprotein and its specific activity is 391.9 U/mg.

Cloning and sequence analysis of a novel phenylalanine ammonia-lyase gene from Astragalus membranaceus / 中草药

Objective To clone and sequence the cDNA encoding phenylalanine ammonia-lyase(PAL)gene from Astragalus membranaceus.Methods RT-PCR and RACE Techniques were used to clone a phenylalanine ammonia-lyase gene from A.membranaceus roots with the total RNA as the template.Results The cloned gene named as AmPAL and the Genbank registry number is EF567076.Squence analysis showed that the full-length of AmPAL cDNA was 2 650 bp,including a 2 154 bp open reading frame(ORF).AmPAL was a new number of PAL family that consisted of 718 amino acids with prediated mole-cular weight of 7.805?104 and isoelectric point(PI)of 5.96.At the same time,AmPAL had the homo-logy with PAL of known leguminous plants and shared above 80% identity of amino acid sequences.Conclusion It is the first report that a novel PAL gene is cloned from A.membranaceus.This work lays a foundation for regulating phenylpropanoid pathway of medical plant with AmPAL.

Flavonoid constituents of Astragalus membranaceus var. mongholicus / 中草药

Objective To study the chemical constituents of Astragalus membranaceus var. mongholicus. Methods The constituents were isolated and purified by several chromatographic techniques and identified by chemicophysical properties and spectral analyses. Results Nine flavonoid compounds had been obtained from A. membranaceus var. mongholicus. They were determined as formononetin (Ⅰ), (3R)-8, 2′-dihydroxy-7, 4′-dimethoxy-isoflavane (Ⅱ), calycosin (Ⅲ), (6aR, 11aR)9, 10-dimethoxypterocarpan-3-O-?-D-glucoside (Ⅳ), 7, 2′-dihydroxy-3′, 4′-dimethoxy-isoflavane-7-O-?-D-glucoside (Ⅴ), formononetin-7-O-?-D-glucoside (Ⅵ), calycosin-7-O-?-D-glucoside (Ⅶ), pratensein-7-O-?-D-glucoside (Ⅷ), and genistin (Ⅸ), respectively. Conclusion Compound Ⅷ is obtained from the plants of Astragalus Linn. for the first time and compound Ⅱ is obtained from this plant for the first time. Compounds Ⅰ-Ⅶ show cell multiplication activity.

Morphologic characteristics and identification methods of seeds in Astragalus membranaceus var.mongholicus and A.membranaceus / 中草药

Objective To provide a method for identifying the seeds of Astragalus membranaceus var.mongholicus(AMM) and A.membranaceus(AM) and to provide basic research data for establishing relevant Standard Operating Practice(SOP) in accordance with GAP of traditional Chinese medicinal materials.Methods The seed morphologic characteristics and microcosmic structures were observed by eyes,light microscopes,and electron microscopes;the seed germination rates of AMM and AM were also compared.Results There was no obvious discrimination of the seed morphology between AMM and AM.There was obvious discrimination on the characteristic of germination-hole,the microcosmic structures of seed-umbilici and seed-coats of seeds between AMM and AM.The hard seed percentage for AMM was higher than that of AM,and its sprouting was not even and sprouting peak appeared later than that of AM.(Conclusion) The seeds of AMM and AM can be identified accuratly with electron microscopes.The patterns of germination-hole,microcosmic structures of seed-umbilici and seed-coats can be used as indices to identify the seeds of AMM and AM.The hard seed percentage and germination characteristics can be used to(identify) the seeds of AMM and AM subsidiarily.