The effects of three different grinding methods in DNA extraction of cowpea (Vigna unguiculata L. Walp)

Rapid DNA extraction is a prerequisite for molecular studies. Generally, plant tissue is ground in liquid nitrogen to isolate DNA; but, liquid nitrogen is dangerous and volatile. Besides, liquid nitrogen is not always available in many developing countries. To investigate if high quality DNA could be obtained for downstream PCR analysis without liquid nitrogen, the cowpea DNA was extracted by Hexadecyl trimethyl ammonium bromide cetyl trimethylammonium bromide (CTAB) method and sodium dodecyl sulphate (SDS) method, respectively, each with three different grinding methods, including ground in liquid nitrogen, in preheated mortar and in non-preheated mortar. The DNA was compared according to their yield, purity, integrity and functionality. The results showed that high quality DNA could be obtained by three grinding methods both in CTAB method and SDS method. Without liquid nitrogen, grinding plant tissue in preheated or non-preheated mortar with extraction buffer to extract DNA is feasible.Keywords: Cowpea (Vigna unguiculata), grinding method, liquid nitrogen, DNA extractionAfrican Journal of Biotechnology Vol. 12(16), pp. 1946-195

Genome and pan-genome assembly of asparagus bean (Vigna unguiculata ssp. sesquipedialis) reveal the genetic basis of cold adaptation

Asparagus bean (Vigna unguiculata ssp. sesquipedialis) is an important cowpea subspecies. We assembled the genomes of Ningjiang 3 (NJ, 550.31 Mb) and Dubai bean (DB, 564.12 Mb) for comparative genomics analysis. The whole-genome duplication events of DB and NJ occurred at 64.55 and 64.81 Mya, respectively, while the divergence between soybean and Vigna occurred in the Paleogene period. NJ genes underwent positive selection and amplification in response to temperature and abiotic stress. In species-specific gene families, NJ is mainly enriched in response to abiotic stress, while DB is primarily enriched in respiration and photosynthesis. We established the pan-genomes of four accessions (NJ, DB, IT97K-499-35 and Xiabao II) and identified 20,336 (70.5%) core genes present in all the accessions, 6,507 (55.56%) variable genes in two individuals, and 2,004 (6.95%) unique genes. The final pan genome is 616.35 Mb, and the core genome is 399.78 Mb. The variable genes are manifested mainly in stress response functions, ABC transporters, seed storage, and dormancy control. In the pan-genome sequence variation analysis, genes affected by presence/absence variants were enriched in biological processes associated with defense responses, immune system processes, signal transduction, and agronomic traits. The results of the present study provide genetic data that could facilitate efficient asparagus bean genetic improvement, especially in producing cold-adapted asparagus bean

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature

A bibliometric analysis in gene research of myocardial infarction from 2001 to 2015

Objectives We aimed to evaluate the global scientific output of gene research of myocardial infarction and explore their hotspots and frontiers from 2001 to 2015, using bibliometric methods. Methods Articles about the gene research of myocardial infarction between 2001 and 2015 were retrieved from the Web of Science Core Collection (WoSCC). We used the bibliometric method and Citespace V to analyze publication years, journals, countries, institutions, research areas, authors, research hotspots, and trends. We plotted the reference co-citation network, and we used key words to analyze the research hotspots and trends. Results We identified 1,853 publications on gene research of myocardial research from 2001 to 2015, and the annual publication number increased with time. Circulation published the highest number of articles. United States ranked highest in the countries with most publications, and the leading institute was Harvard University. Relevant publications were mainly in the field of Cardiovascular system cardiology. Keywords and references analysis indicated that gene expression, microRNA and young women were the research hotspots, whereas stem cell, chemokine, inflammation and cardiac repair were the frontiers. Conclusions We depicted gene research of myocardial infarction overall by bibliometric analysis. Mesenchymal stem cells Therapy, MSCs-derived microRNA and genetic modified MSCs are the latest research frontiers. Related studies may pioneer the future direction of this filed in next few years. Further studies are needed

Transcriptome Profiling of Two Asparagus Bean (Vigna unguiculata subsp. sesquipedalis) Cultivars Differing in Chilling Tolerance under Cold Stress.

Cowpea (V. unguiculata L. Walp.) is an important tropical grain legume. Asparagus bean (V. unguiculata ssp. sesquipedialis) is a distinctive subspecies of cowpea, which is considered one of the top ten Asian vegetables. It can be adapted to a wide range of environmental stimuli such as drought and heat. Nevertheless, it is an extremely cold-sensitive tropical species. Improvement of chilling tolerance in asparagus bean may significantly increase its production and prolong its supply. However, gene regulation and signaling pathways related to cold response in this crop remain unknown. Using Illumina sequencing technology, modification of global gene expression in response to chilling stress in two asparagus bean cultivars-"Dubai bean" and "Ningjiang-3", which are tolerant and sensitive to chilling, respectively-were investigated. More than 1.8 million clean reads were obtained from each sample. After de novo assembly, 88,869 unigenes were finally generated with a mean length of 635 bp. Of these unigenes, 41,925 (47.18%) had functional annotations when aligned to public protein databases. Further, we identified 3,510 differentially expressed genes (DEGs) in Dubai bean, including 2,103 up-regulated genes and 1,407 down-regulated genes. While in Ningjiang-3, we found 2,868 DEGs, 1,786 of which were increasing and the others were decreasing. 1,744 DEGs were commonly regulated in two cultivars, suggesting that some genes play fundamental roles in asparagus bean during cold stress. Functional classification of the DEGs in two cultivars using Mercator pipeline indicated that RNA, protein, signaling, stress and hormone metabolism were five major groups. In RNA group, analysis of TFs in DREB subfamily showed that ICE1-CBF3-COR cold responsive cascade may also exist in asparagus bean. Our study is the first to provide the transcriptome sequence resource for asparagus bean, which will accelerate breeding cold resistant asparagus bean varieties through genetic engineering, and advance our knowledge of the genes involved in the complex regulatory networks of this plant under cold stress

Transcriptome analysis of green and purple fruited pepper provides insight into novel regulatory genes in anthocyanin biosynthesis

Background Pepper (Capsicum annuum L.) is a valuable horticultural crop with economic significance, and its purple fruit color is attributed to anthocyanin, a phytonutrient known for its health-promoting benefits. However, the mechanisms regulating anthocyanin biosynthesis in pepper have yet to be fully elucidated. Methods RNA sequencing (RNA-seq) was utilized to analyze the transcriptome of fruits from three purple-fruited varieties (HN191, HN192, and HN005) and one green-fruited variety (EJT) at various developmental stages. To determine the relationships between samples, Pearson correlation coefficients (PCC) and principal component analysis (PCA) were calculated. Differential expression analysis was performed using the DESeq2 package to identify genes that were expressed differently between two samples. Transcription factors (TF) were predicted using the iTAK program. Heatmaps of selected genes were generated using Tbtools software. Results The unripe fruits of HN191, HN192, and HN005, at the stages of 10, 20, and 30 days after anthesis (DAA), display a purple color, whereas the unripe fruits of variety EJT remain green. To understand the molecular basis of this color difference, five transcriptome comparisons between green and purple fruits were conducted: HN191-10 vs EJT-10, HN191-20 vs EJT-20, HN191-30 vs EJT-30, HN192-30 vs EJT-30, and HN005-30 vs EJT-30. Through this analysis, 503 common differentially expressed genes (DEGs) were identified. Among these DEGs, eight structural genes related to the anthocyanin biosynthesis pathway and 24 transcription factors (TFs) were detected. Notably, one structural gene (MSTRG.12525) and three TFs (T459_25295, T459_06113, T459_26036) exhibited expression patterns that suggest they may be novel candidate genes involved in anthocyanin biosynthesis. These results provide new insights into the regulation of anthocyanin biosynthesis in purple pepper fruit and suggest potential candidate genes for future genetic improvement of pepper germplasm with enhanced anthocyanin accumulation

A 16-gene signature predicting prognosis of patients with oral tongue squamous cell carcinoma

Background Oral tongue squamous cell carcinoma (OTSCC) is the most common subtype of oral cancer. A predictive gene signature is necessary for prognosis of OTSCC. Methods Five microarray data sets of OTSCC from the Gene Expression Omnibus (GEO) and one data set from The Cancer Genome Atlas (TCGA) were obtained. Differentially expressed genes (DEGs) of GEO data sets were identified by integrated analysis. The DEGs associated with prognosis were screened in the TCGA data set by univariate survival analysis to obtain a gene signature. A risk score was calculated as the summation of weighted expression levels with coefficients by Cox analysis. The signature was used to distinguish carcinoma, estimated by receiver operator characteristic curves and the area under the curve (AUC). All were validated in the GEO and TCGA data sets. Results Integrated analysis of GEO data sets revealed 300 DEGs. A 16-gene signature and a risk score were developed after survival analysis. The risk score was effective to stratify patients into high-risk and low-risk groups in the TCGA data set (P < 0.001). The 16-gene signature was valid to distinguish the carcinoma from normal samples (AUC 0.872, P < 0.001). Discussion We identified a useful 16-gene signature for prognosis of OTSCC patients, which could be applied to clinical practice. Further studies were needed to prove the findings

Pig Coat Color Manipulation by <i>MC1R</i> Gene Editing

Black coat color in pigs is determined by the dominant E allele at the MC1R locus. Through comparing MC1R gene sequences between recessive e and dominant ED1 alleles, we identified four missense mutations that could affect MC1R protein function for eumelanin synthesis. With the aim of devising a genetic modification method for pig coat color manipulation, we mutated the e allele in the Duroc breed to the dominant ED1 allele using CRISPR-mediated homologous recombination for the four mutation substitutions at the MC1R locus. The MC1R-modified Duroc pigs generated using the allele replacement strategy displayed uniform black coat color across the body. A genotyping assay showed that the MC1R-modified Duroc pigs had a heterozygous ED1/e allele at the MC1R locus; in addition, the pigs remained in the Duroc genetic background. Our work offers a gene editing method for pig coat color manipulation, which could value the culture of new pig varieties meeting the needs of diversified market

Estimating 3D Green Volume and Aboveground Biomass of Urban Forest Trees by UAV-Lidar

Three dimensional (3D) green volume is an important tree factor used in forest surveys as a prerequisite for estimating aboveground biomass (AGB). In this study, we developed a method for accurately calculating the 3D green volume of single trees from unmanned aerial vehicle laser scanner (ULS) data, using a voxel coupling convex hull by slices algorithm, and compared the results using voxel coupling convex hull by slices algorithm with traditional 3D green volume algorithms (3D convex hull, 3D concave hull (alpha shape), convex hull by slices, voxel and voxel coupling convex hull by slices algorithms) to estimate AGB. Our results showed the following: (1) The voxel coupling convex hull by slices algorithm can accurately estimate the 3D green volume of a single ginkgo tree (RMSE = 11.17 m3); (2) Point cloud density can significantly affect the extraction of 3D green volume; (3) The addition of the 3D green volume parameter can significantly improve the accuracy of the model to estimate AGB, where the highest accuracy was obtained by the voxel coupling convex hull by slices algorithm (CV-R2 = 0.85, RMSE = 11.29 kg, and nRMSE = 15.12%). These results indicate that the voxel coupling convex hull by slices algorithms can more effectively calculate the 3D green volume of a single tree from ULS data. Moreover, our study provides a comprehensive evaluation of the use of ULS 3D green volume for AGB estimation and could significantly improve the estimation accuracy of AGB