Flower of Abelmoschus manihot (FAM) is clinically effective to treat chronic kidney disease (CKD) with a relatively high dosage. To improve the efficacy and the compliance of patients, macroporous resins were adopted to enrich and purify flavonoids from FAM, which are thought to be the major renal protective constituents in FAM. After screening six different kinds of macroporous resins, HPD-100 was selected for its great adsorption and desorption capacity. Then, orthogonal design tests were used to optimize parameters in the processes of impurity removal and flavonoids of FAM desorption on column chromatogram. Moreover, process scale-up was performed, and purification effects maintained after amplification. After purification, the content of seven main flavonoids in the product increased from 8.29% to 51.43%. Protective and anti-inflammatory effects of crude extract and the flavonoid component of FAM after purification were investigated on the adriamycin-damaged HK-2 cells and lipopolysaccharide-stimulated Raw 264.7 cells models. Both bioactivities were improved greatly after purification for these two cell models. Therefore, the purification process had enriched the main bioactive constituents with potential alleviating kidney injury activities. The flavonoid component of FAM is worthy of being developed as an improved remedy for CKD with better patients’ compliance.

目的：分析不同来源的黄蜀葵胶中的糖含量、多糖相对分子质量及其单糖组成。方法：采用3,5-二硝基水杨酸比色法测定4种黄蜀葵胶中还原糖、总糖和总多糖的含量，并解析其红外吸收光谱图；凝胶渗透色谱法测定纯化后的4种多糖的相对分子质量，气相色谱法分析其单糖组成。结果：4种黄蜀葵胶的还原糖质量分数分别为51.07%、9.00%、53.94%和11.46%，总糖质量分数分别为79.95%、78.99%、91.98%和61.13%，总多糖质量分数分别为28.88%、69.99%、38.04%和49.67%；红外谱图显示AMG-1、AMG-3中主要含α-D-吡喃葡萄糖，AMG-2和AMG-4中主要含有甘露糖；4种黄蜀葵胶中多糖的峰值相对分子质量分别为3.91×103、5.36×105、3.87×103和5.12×105；4种样品均含有甘露糖、葡萄糖和半乳糖，其物质量之比分别为0.42:1.00:0.27、1.00:0.15:0.54、0.27:1.00:0.15和1.00:0.12:0.48。结论：不同来源的黄蜀葵胶在还原糖、总糖和总多糖的含量，多糖相对分子质量及其单糖组成上均存在较大差异。

Abelmoschus is an economically and phylogenetically valuable genus in the family Malvaceae. Owing to coexistence of wild and cultivated form and interspecific hybridization, this genus is controversial in systematics and taxonomy and requires detailed investigation. Here, we present whole chloroplast genome sequences and annotation of three important species: A. moschatus, A. manihot and A. sagittifolius, and compared with A. esculentus published previously. These chloroplast genome sequences ranged from 163121 bp to 163453 bp in length and contained 132 genes with 87 protein-coding genes, 37 transfer RNA and 8 ribosomal RNA genes. Comparative analyses revealed that amino acid frequency and codon usage had similarity among four species, while the number of repeat sequences in A. esculentus were much lower than other three species. Six categories of simple sequence repeats (SSRs) were detected, but A. moschatus and A. manihot did not contain hexanucleotide SSRs. Single nucleotide polymorphisms (SNPs) of A/T, T/A and C/T were the largest number type, and the ratio of transition to transversion was from 0.37 to 0.55. Abelmoschus species showed relatively independent inverted-repeats (IR) boundary traits with different boundary genes compared with the other related Malvaceae species. The intergenic spacer regions had more polymorphic than protein-coding regions and intronic regions, and thirty mutational hotpots (≥200 bp) were identified in Abelmoschus, such as start-psbA, atpB-rbcL, petD-exon2-rpoA, clpP-intron1 and clpP-exon2.These mutational hotpots could be used as polymorphic markers to resolve taxonomic discrepancies and biogeographical origin in genus Abelmoschus. Moreover, phylogenetic analysis of 33 Malvaceae species indicated that they were well divided into six subfamilies, and genus Abelmoschus was a well-supported clade within genus Hibiscus.

为从转录水平上解析穿心莲体内主要进行的生物进程及其分子机制,选取生长60天的福建漳州生产用穿心莲苗为材料,利用超长单分子测序技术测得其根、茎和倒三叶的全长转录组初始序列信息。对初始序列进行筛选、去冗余、校正、功能注释和基因结构分析。结果显示,穿心莲基因的功能主要富集于代谢途径、次生代谢产物合成途径及抗生素合成途径。bHLH、bZIP、MYB和WRKY等直接参与次生代谢的转录因子含量位居前10。穿心莲内酯前体合成途径基因出现可变剪切,主要为内含子保留。其中,有1个基因产生了6个可变启动子式的内含子保留mRNA亚型。总之,次生代谢是穿心莲的主要生物活性进程,相关功能基因可通过结合丰富的转录因子和进行高频的可变剪切参与其中。

We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process.Blast2GO is freely available via Java Web Start at http://www.blast2go.de.http://www.blast2go.de -> Evaluation.

In the process of making full-length cDNA, predicting protein coding regions helps both in the preliminary analysis of genes and in any succeeding process. However, unfinished cDNA contains artifacts including many sequencing errors, which hinder the correct evaluation of coding sequences. Especially, predictions of short sequences are difficult because they provide little information for evaluating coding potential. In this paper, we describe ANGLE, a new program for predicting coding sequences in low quality cDNA. To achieve error-tolerant prediction, ANGLE uses a machine-learning approach, which makes better expression of coding sequence maximizing the use of limited information from input sequences. Our method utilizes not only codon usage, but also protein structure information which is difficult to be used for stochastic model-based algorithms, and optimizes limited information from a short segment when deciding coding potential, with the result that predictive accuracy does not depend on the length of an input sequence. The performance of ANGLE is compared with ESTSCAN on four dataset each of them having a different error rate (one frame-shift error or one substitution error per 200-500 nucleotides) and on one dataset which has no error. ANGLE outperforms ESTSCAN by 9.26% in average Matthews's correlation coefficient on short sequence dataset (< 1000 bases). On long sequence dataset, ANGLE achieves comparable performance.

Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available data sets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA-sequencing data compared with experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large data sets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa. © 2015 Alamancos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-β-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-β-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.

为了深入了解甘葛藤转录组的整体水平及黄酮类生物合成通路基因。利用高通量测序PacBio Sequel平台，以甘葛藤根、茎、叶的混合样品为材料，使用单分子长读数测序技术（SMRT）对甘葛藤进行全长转录组测序及分析。平台共获得10 994 967个高质量reads和384 072条全长非嵌合序列（FLNC），测序数据经质控后获得90 856个转录本；获得的所有转录本经NR、SwissProt、KOG、KEGG、GO数据库进行注释和功能分类，结果有85 239个单基因被注释，NR注释数量最多为84 675个，占93.2%；KEGG注释的基因最少，22 330个基因被注释到132条途径，代谢途径分布的基因较多（9 368，41.95%）。预测到3 507个转录因子，bHLH转录因子家族的基因最多。14 127个基因被分配到17个R基因类别，主要为RLP类。检测到33 660个SSR序列，多为AG/CT类型。分析黄酮类生物合成途径，发现与黄酮类合成相关的基因110个，其中，26个编码HCT，3个编码CHS，7个编码CHI。PacBio测序平台能获得更长的转录本，SMRT技术能够深入挖掘甘葛藤转录数据，比第二代测序技术能够获得更高的转录本注释率。在高通量全长转录组水平对甘葛藤进行了研究，为甘葛藤的分子生物学研究提供了较可靠、全面的转录组数据，为进一步开发甘葛藤的分子标记和挖掘优良基因提供了科学依据。