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https://www.readbyqxmd.com/read/28646085/generation-of-a-collection-of-mutant-tomato-lines-using-pooled-crispr-libraries
#1
Thomas B Jacobs, Ning Zhang, Dhruv Patel, Gregory B Martin
The high efficiency of CRIPSR-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato, collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated gRNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted...
June 23, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28638397/generation-annotation-and-analysis-of-a-large-scale-expressed-sequence-tag-library-from-arabidopsis-pumila-to-explore-salt-responsive-genes
#2
Xianzhong Huang, Lifei Yang, Yuhuan Jin, Jun Lin, Fang Liu
Arabidopsis pumila is an ephemeral plant, and a close relative of the model plant Arabidopsis thaliana, but it possesses higher photosynthetic efficiency, higher propagation rate, and higher salinity tolerance compared to those A. thaliana, thus providing a candidate plant system for gene mining for environmental adaption and salt tolerance. However, A. pumila is an under-explored resource for understanding the genetic mechanisms underlying abiotic stress adaptation. To improve our understanding of the molecular and genetic mechanisms of salt stress adaptation, more than 19,900 clones randomly selected from a cDNA library constructed previously from leaf tissue exposed to high-salinity shock were sequenced...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28634080/nong-a-constituent-of-the-nonactin-biosynthetic-gene-cluster-regulates-nocardamine-synthesis-in-streptomyces-albus-j1074
#3
Wanki Park, Jung-Kyun Woo, Jongheon Shin, Ki-Bong Oh
Many factors regulate the expression of specialised secondary metabolite biosynthetic gene clusters, which have been recognised as important for the discovery of novel microbial natural products. A cosmid library based on genomic DNA of the marine-derived Streptomyces puniceus Act1085 was constructed and screened to identify a short gene cluster similar to the nonactin biosynthetic cluster. The ORFs of the gene cluster isolated had high amino acid sequence identity, from 82% to 96%, with corresponding ORFs of the nonactin biosynthetic gene cluster from S...
June 17, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28630098/comparative-micrornaome-analysis-of-the-testis-and-ovary-of-the-chinese-giant-salamander
#4
Rui Chen, Jian Du, Lin Ma, Li-Qing Wang, Sheng-Song Xie, Chang-Ming Yang, Xian-Yong Lan, Chuan-Ying Pan, Wu-Zi Dong
MicroRNAs (miRNAs) are 18-24 nucleotides non-coding RNAs that regulate gene expression by post-transcriptional suppression of mRNA. The Chinese giant salamander (CGS, Andrias davidianus), which is an endangered species, has become one of important models of animal evolution; however, no miRNA studies on this species have been conducted. In this study, two small RNA libraries of CGS ovary and testis were constructed using deep sequencing technology. A bioinformatics pipeline was developed to distinguish miRNA sequences from other classes of small RNAs represented in the sequencing data...
June 19, 2017: Reproduction: the Official Journal of the Society for the Study of Fertility
https://www.readbyqxmd.com/read/28622855/b-cell-cross-epitope-of-propionibacterium-acnes-and-actinobacillus-pleuropneumonia-selected-by-phage-display-library-can-efficiently-protect-from-actinobacillus-pleuropneumonia-infection
#5
Jianfang Liu, Qiuyue Ma, Feng Yang, Rining Zhu, Jingmin Gu, Changjiang Sun, Xin Feng, Chongtao Du, Paul R Langford, Wenyu Han, Junling Yang, Liancheng Lei
Contagious porcine pleuropneumonia (CPP), caused by Actinobacillus pleuropneumoniae (APP), is a highly transmissible and fatal respiratory illness that causes tremendous economic losses for the pig breeding industry worldwide. Propionibacterium acnes (PA) has a strong cross-reaction with anti-APP1 and anti-APP5 serum and can efficiently prevent APP infection, which was fortuitously found in researching the differential gene between the different APP serotypes. There seems to be some natural cross-protection between PA and APP...
June 2017: Veterinary Microbiology
https://www.readbyqxmd.com/read/28622611/de-novo-transcriptome-sequencing-and-comparative-analysis-to-discover-genes-involved-in-ovarian-maturity-in-strongylocentrotus-nudus
#6
Zhiying Jia, Qiai Wang, Kaikai Wu, Zhenlin Wei, Zunchun Zhou, Xiaolin Liu
Strongylocentrotus nudus is an edible sea urchin, mainly harvested in China. Correlation studies indicated that S. nudus with larger diameter have a prolonged marketing time and better palatability owing to their precocious gonads and extended maturation process. However, the molecular mechanism underlying this phenomenon is still unknown. Here, transcriptome sequencing was applied to study the ovaries of adult S. nudus with different shell diameters to explore the possible mechanism. In this study, four independent cDNA libraries were constructed, including two from the big size urchins and two from the small ones using a HiSeq™2500 platform...
May 30, 2017: Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics
https://www.readbyqxmd.com/read/28621534/construction-of-an-immunized-rabbit-phage-display-library-for-selecting-high-activity-of-against-bacillus-thuringiensis-cry1f-toxin-single-chain-antibodies
#7
Chongxin Xu, Cunzheng Zhang, Jianfeng Zhong, Hui Hu, Shimin Luo, Xiaoqin Liu, Xiao Zhang, Yuan Liu, Xianjin Liu
In the present study, a Cry1F-immunized rabbit phage display library (6.96×108) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (scFv) by bio-panning. Total 16 positive monoclonal phage scFvs were obtained after 4 rounds panning which identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing. The most positive phage scFv (named RF4) was expressed in E.coli.HB2151 and the purified soluble protein approximate 30 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)...
June 16, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28613262/antimicrobial-activity-of-truncated-and-polyvalent-peptides-derived-from-the-fkcrrqwqwrmkkgla-sequence-against-escherichia-coli-atcc-25922-and-staphylococcus-aureus-atcc-25923
#8
Nataly de Jesús Huertas, Zuly Jenny Rivera Monroy, Ricardo Fierro Medina, Javier Eduardo García Castañeda
Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Specifically, a peptide library was constructed by systemically removing the flanking residues (N or C-terminal) of Lfcin 17-31 ((17)FKCRRWQWRMKKLGA(31)), maintaining in all peptides the (20)RRWQWR(25) sequence that corresponds to the minimal antimicrobial motif. For this research, also included were (i) a peptide containing an Ala instead of Cys ([Ala(19)]-LfcinB 17-31) and (ii) polyvalent peptides containing the RRWQWR sequence and a non-natural amino acid (aminocaproic acid)...
June 14, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/28611392/targeted-sequencing-of-both-dna-strands-barcoded-and-captured-individually-by-rna-probes-to-identify-genome-wide-ultra-rare-mutations
#9
Qing Wang, Xu Wang, Pheobe S Tang, Grace M O'leary, Ming Zhang
Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq)...
June 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28609783/balancing-gene-expression-without-library-construction-via-a-reusable-srna-pool
#10
Amar Ghodasara, Christopher A Voigt
Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ∼103-fold range of strengths, yielding ∼107 combinations...
June 13, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28606864/suppression-of-nf-%C3%AE%C2%BAb-signal-pathway-by-nlrc3-like-protein-in-stony-coral-acropora-aculeus-under-heat-stress
#11
Zhi Zhou, Yibo Wu, Chengkai Zhang, Can Li, Guangmei Chen, Xiaopeng Yu, Xiaowei Shi, Yanlai Xu, Lingui Wang, Bo Huang
Heat stress is the most common factor for coral bleaching, which has increased both in frequency and severity due to global warming. In the present study, the stony coral Acropora aculeus was subjected to acute heat stress and entire transcriptomes were sequenced via the next generation sequencing platform. Four paired-end libraries were constructed and sequenced in two groups, including a control and a heat stress group. A total of 120,319,751 paired-end reads with lengths of 2 × 100 bp were assembled and 55,021 coral-derived genes were obtained...
June 9, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28605506/multi-locus-and-long-amplicon-sequencing-approach-to-study-microbial-diversity-at-species-level-using-the-miniontm-portable-nanopore-sequencer
#12
Alfonso Benítez-Páez, Yolanda Sanz
Background: The miniaturised and portable DNA sequencer MinION TM has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION TM platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. Results: After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67,000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION TM device using R9 and R9...
June 12, 2017: GigaScience
https://www.readbyqxmd.com/read/28603873/environmental-dna-enables-detection-of-terrestrial-mammals-from-forest-pond-water
#13
Masayuki Ushio, Hisato Fukuda, Toshiki Inoue, Kobayashi Makoto, Osamu Kishida, Keiichi Sato, Koichi Murata, Masato Nikaido, Tetsuya Sado, Yukuto Sato, Masamichi Takeshita, Wataru Iwasaki, Hiroki Yamanaka, Michio Kondoh, Masaki Miya
Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues...
June 11, 2017: Molecular Ecology Resources
https://www.readbyqxmd.com/read/28602759/construction-and-screening-of-a-lentiviral-secretome-library
#14
Tao Liu, Panpan Jia, Huailei Ma, Sean A Reed, Xiaozhou Luo, H Benjamin Larman, Peter G Schultz
Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77...
June 22, 2017: Cell Chemical Biology
https://www.readbyqxmd.com/read/28598995/plant-cell-wall-glycosyltransferases-high-throughput-recombinant-expression-screening-and-general-requirements-for-these-challenging-enzymes
#15
Ditte Hededam Welner, David Shin, Giovani P Tomaleri, Andy M DeGiovanni, Alex Yi-Lin Tsai, Huu M Tran, Sara Fasmer Hansen, Derek T Green, Henrik V Scheller, Paul D Adams
Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability...
2017: PloS One
https://www.readbyqxmd.com/read/28596758/a-functional-metagenomic-analysis-of-tetracycline-resistance-in-cheese-bacteria
#16
Ana B Flórez, Lucía Vázquez, Baltasar Mayo
Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28593475/functional-metagenomics-of-oil-impacted-mangrove-sediments-reveals-high-abundance-of-hydrolases-of-biotechnological-interest
#17
Júlia Ronzella Ottoni, Lucélia Cabral, Sanderson Tarciso Pereira de Sousa, Gileno Vieira Lacerda Júnior, Daniela Ferreira Domingos, Fábio Lino Soares Junior, Mylenne Calciolari Pinheiro da Silva, Joelma Marcon, Armando Cavalcante Franco Dias, Itamar Soares de Melo, Anete Pereira de Souza, Fernando Dini Andreote, Valéria Maia de Oliveira
Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms...
July 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28582444/selection-and-characterization-of-specific-nanobody-against-bovine-virus-diarrhea-virus-bvdv-e2-protein
#18
Tiansen Li, Meiling Huang, Hongran Xiao, Guoqi Zhang, Jinhua Ding, Peng Wu, Hui Zhang, Jinliang Sheng, Chuangfu Chen
Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank...
2017: PloS One
https://www.readbyqxmd.com/read/28572808/transcriptome-wide-analysis-of-botrytis-elliptica-responsive-micrornas-and-their-targets-in-lilium-regale-wilson-by-high-throughput-sequencing-and-degradome-analysis
#19
Xue Gao, Qi Cui, Qin-Zheng Cao, Qiang Liu, Heng-Bin He, Dong-Mei Zhang, Gui-Xia Jia
MicroRNAs, as master regulators of gene expression, have been widely identified and play crucial roles in plant-pathogen interactions. A fatal pathogen, Botrytis elliptica, causes the serious folia disease of lily, which reduces production because of the high susceptibility of most cultivated species. However, the miRNAs related to Botrytis infection of lily, and the miRNA-mediated gene regulatory networks providing resistance to B. elliptica in lily remain largely unexplored. To systematically dissect B. elliptica-responsive miRNAs and their target genes, three small RNA libraries were constructed from the leaves of Lilium regale, a promising Chinese wild Lilium species, which had been subjected to mock B...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28570594/automated-high-throughput-nucleic-acid-purification-from-formalin-fixed-paraffin-embedded-tissue-samples-for-next-generation-sequence-analysis
#20
Simon Haile, Pawan Pandoh, Helen McDonald, Richard D Corbett, Philip Tsao, Heather Kirk, Tina MacLeod, Martin Jones, Steve Bilobram, Denise Brooks, Duane Smailus, Christian Steidl, David W Scott, Miruna Bala, Martin Hirst, Diane Miller, Richard A Moore, Andrew J Mungall, Robin J Coope, Yussanne Ma, Yongjun Zhao, Rob A Holt, Steven J Jones, Marco A Marra
Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification...
2017: PloS One
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