Optimization of RAPD-PCR reaction system for genetic relationships analysis of 15 camellia cultivars

With orthogonal analysis by L27(36), the random amplified polymorphic DNA (RAPD)-PCR optimization reaction system for camellia were obtained. Results showed that the optimization system was 10×PCR Buffer (2.5 L), 25 mM MgCl2 (2.5 L), 2.5 mM dNTPs (2.0 L), 20 M primer (1.0 L), Tag (1.5 U), temple DNA (40 ng or so) and added ddH2O to the total volume 25 uL; suitable annealing temperature was 36°C. With the optimized system and fifteen 10 nt random primers, we analyzed 15 camellia cultivars and observed 102 clear amplified loci, in which polymorphic loci were 79 while the percentage of polymorphic loci were 77.54%. Cluster analysis showed that the four groups were divided at the point 0.75 of similarity coefficient, indicating relatively high genetic diversity. We also found that the gene controlling petal color may play an important role in RAPD analysis. Moreover, genetic diversities based on RAPD analysis could be clearly reflected by morphological traits among 15 camellia cultivars. This study showed the RAPD optimization system was suitable and RAPD molecular marker was effective and useful tool for detection of genetic relationships among camellia cultivars

β-Elemene-induced autophagy protects human gastric cancer cells from undergoing apoptosis

<p>Abstract</p> <p>Background</p> <p>β-Elemene, a compound found in an herb used in traditional Chinese medicine, has shown promising anti-cancer effects against a broad spectrum of tumors. The mechanism by which β-elemene kills cells remains unclear. The aim of the present study is to investigate the anti-tumor effect of β-elemene on human gastric cancer cells and the molecular mechanism involved.</p> <p>Results</p> <p>β-Elemene inhibited the viability of human gastric cancer MGC803 and SGC7901 cells in a dose-dependent manner. The suppression of cell viability was due to the induction of apoptosis. A robust autophagy was observed in the cells treated with β-elemene; it was characterized by the increase of punctate LC3 dots, the cellular morphology, and the increased levels of LC3-II protein. Further study showed that β-elemene treatment up-regulated Atg5-Atg12 conjugated protein but had little effect on other autophagy-related proteins. PI3K/Akt/mTOR/p70S6K1 activity was inhibited by β-elemene. Knockdown of Beclin 1 with small interfering RNA, or co-treatment with the autophagy inhibitor, 3-methyladenine or chlorochine enhanced significantly the antitumor effects of β-elemene.</p> <p>Conclusions</p> <p>Our data provides the first evidence that β-elemene induces protective autophagy and prevents human gastric cancer cells from undergoing apoptosis. A combination of β-elemene with autophagy inhibitor might thus be a useful therapeutic option for advanced gastric cancer.</p

A cotton miRNA is involved in regulation of plant response to salt stress

The present study functionally identified a new microRNA (microRNA ovual line 5, miRNVL5) with its target gene GhCHR from cotton (Gossypium hirsutum). The sequence of miRNVL5 precursor is 104 nt long, with a well developed secondary structure. GhCHR contains two DC1 and three PHD Cys/His-rich domains, suggesting that GhCHR encodes a zinc-finger domain-containing transcription factor. miRNVL5 and GhCHR express at various developmental stages of cotton. Under salt stress (50–400 mM NaCl), miRNVL5 expression was repressed, with concomitant high expression of GhCHR in cotton seedlings. Ectopic expression of GhCHR in Arabidopsis conferred salt stress tolerance by reducing Na+ accumulation in plants and improving primary root growth and biomass. Interestingly, Arabidopsis constitutively expressing miRNVL5 showed hypersensitivity to salt stress. A GhCHR orthorlous gene At2g44380 from Arabidopsis that can be cleaved by miRNVL5 was identified by degradome sequencing, but no confidential miRNVL5 homologs in Arabidopsis have been identified. Microarray analysis of miRNVL5 transgenic Arabidopsis showed six downstream genes (CBF1, CBF2, CBF3, ERF4, AT3G22920, and AT3G49200), which were induced by salt stress in wild-type but repressed in miRNVL5-expressing Arabidopsis. These results indicate that miRNVL5 is involved in regulation of plant response to salt stress

MicroRNA expression profiles during cotton (Gossypium hirsutum L) fiber early development

The role of microRNAs (miRNAs) during cotton fiber development remains unclear. Here, a total of 54 miRNAs belonging to 39 families were selected to characterize miRNA regulatory mechanism in eight different fiber development stages in upland cotton cv BM-1. Among 54 miRNAs, 18 miRNAs were involved in cotton fiber initiation and eight miRNAs were related to fiber elongation and secondary wall biosynthesis. Additionally, 3,576 protein-coding genes were candidate target genes of these miRNAs, which are potentially involved in cotton fiber development. We also investigated the regulatory network of miRNAs and corresponding targets in fiber initiation and elongation, and secondary wall formation. Our Gene Ontology-based term classification and KEGG-based pathway enrichment analyses showed that the miRNA targets covered 220 biological processes, 67 molecular functions, 45 cellular components, and 10 KEGG pathways. Three of ten KEGG pathways were involved in lignan synthesis, cell elongation, and fatty acid biosynthesis, all of which have important roles in fiber development. Overall, our study shows the potential regulatory roles of miRNAs in cotton fiber development and the importance of miRNAs in regulating different cell types. This is helpful to design miRNA-based biotechnology for improving fiber quality and yield

Application of Consensus Scoring and Principal Component Analysis for Virtual Screening against β-Secretase (BACE-1)

BACKGROUND: In order to identify novel chemical classes of β-secretase (BACE-1) inhibitors, an alternative scoring protocol, Principal Component Analysis (PCA), was proposed to summarize most of the information from the original scoring functions and re-rank the results from the virtual screening against BACE-1. METHOD: Given a training set (50 BACE-1 inhibitors and 9950 inactive diverse compounds), three rank-based virtual screening methods, individual scoring, conventional consensus scoring and PCA, were judged by the hit number in the top 1% of the ranked list. The docking poses were generated by Surflex, five scoring functions (Surflex_Score, D_Score, G_Score, ChemScore, and PMF_Score) were used for pose extraction. For each pose group, twelve scoring functions (Surflex_Score, D_Score, G_Score, ChemScore, PMF_Score, LigScore1, LigScore2, PLP1, PLP2, jain, Ludi_1, and Ludi_2) were used for the pose rank. For a test set, 113,228 chemical compounds (Sigma-Aldrich® corporate chemical directory) were docked by Surflex, then ranked by the same three ranking methods motioned above to select the potential active compounds for experimental test. RESULTS: For the training set, the PCA approach yielded consistently superior rankings compared to conventional consensus scoring and single scoring. For the test set, the top 20 compounds according to conventional consensus scoring were experimentally tested, no inhibitor was found. Then, we relied on PCA scoring protocol to test another different top 20 compounds and two low micromolar inhibitors (S450588 and 276065) were emerged through the BACE-1 fluorescence resonance energy transfer (FRET) assay. CONCLUSION: The PCA method extends the conventional consensus scoring in a quantitative statistical manner and would appear to have considerable potential for chemical screening applications

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

<p>Abstract</p> <p>Background</p> <p><it>Bupleurum chinense </it>DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of <it>B. chinense</it>, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.</p> <p>Results</p> <p>One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A <it>de novo </it>assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the <it>Bupleurum </it>genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel <it>Bupleurum </it>genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (<it>P450</it>s) and 102 glycosyltransferases (<it>GT</it>s) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 <it>P450</it>s and 7 uridine diphosphate <it>GT</it>s (<it>UGT</it>s) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two <it>P450</it>s and three <it>UGT</it>s were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with <it>β-AS </it>in methyl jasmonate-treated adventitious roots and on their similar expression patterns with <it>β-AS </it>in various <it>B. chinense </it>tissues.</p> <p>Conclusions</p> <p>A collection of high-quality ESTs for <it>B. chinense </it>obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of <it>B. chinense </it>and other <it>Bupleurum </it>species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the <it>P450</it>s and <it>UGT</it>s, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.</p