An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 50% of Drosophila genes lack suitable coding introns for integration of artificial exons. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

Complete Genome Sequence of Bacteriophage EFC1 Infecting Enterococcus Faecalis From Chicken

In response to Enterococcus faecalis infection of chicken origin, a multi host lytic phage, EFC1 was isolated and characterized the double-stranded circular DNA genome with size of 56099 bp, containing 89 predicted protein coding genes as well as 2 tRNAs involved in intron, structure, transcription, packaging, DNA replication, modification, lysis. Observation of the structure by electron microscopy and comparative phylogenetic analysis of terminase large subunit showed that the phage EFC1 belongs to a new member of Siphoviridae, which is relatively distantly related to its high similarity phages. The phage EFC1 has no relevant virulence genes and antibiotic resistance genes.

The origin and evolution of a two-component system of paralogous genes encoding the centromeric histone CENH3 in cereals

Background The cereal family Poaceae is one of the largest and most diverse angiosperm families. The central component of centromere specification and function is the centromere-specific histone H3 (CENH3). Some cereal species (maize, rice) have one copy of the gene encoding this protein, while some (wheat, barley, rye) have two. We applied a homology-based approach to sequenced cereal genomes, in order to finally trace the mutual evolution of the structure of the CENH3 genes and the nearby regions in various tribes. Results We have established that the syntenic group or the CENH3 locus with the CENH3 gene and the boundaries defined by the CDPK2 and bZIP genes first appeared around 50 Mya in a common ancestor of the subfamilies Bambusoideae, Oryzoideae and Pooideae. This locus came to Pooideae with one copy of CENH3 in the most ancient tribes Nardeae and Meliceae. The βCENH3 gene as a part of the locus appeared in the tribes Stipeae and Brachypodieae around 35–40 Mya. The duplication was accompanied by changes in the exon-intron structure. Purifying selection acts mostly on αCENH3s, while βCENH3s form more heterogeneous structures, in which clade-specific amino acid motifs are present. In barley species, the βCENH3 gene assumed an inverted orientation relative to αCENH3 and the CDPK2 gene was substituted with LHCB-l. As the evolution and domestication of plant species went on, the locus was growing in size due to an increasing distance between αCENH3 and βCENH3 because of a massive insertion of the main LTR-containing retrotransposon superfamilies, gypsy and copia, without any evolutionary preference on either of them. A comparison of the molecular structure of the locus in the A, B and D subgenomes of the hexaploid wheat T. aestivum showed that invasion by mobile elements and concomitant rearrangements took place in an independent way even in evolutionarily close species. Conclusions The CENH3 duplication in cereals was accompanied by changes in the exon-intron structure of the βCENH3 paralog. The observed general tendency towards the expansion of the CENH3 locus reveals an amazing diversity of ways in which different species implement the scenario described in this paper.

Genome-Wide Identification and Evolutionary Analysis of the SRO Gene Family in Tomato

SRO (SIMILAR TO RCD ONE) is a family of plant-specific small molecule proteins that play an important role in plant growth and development and environmental responses. However, SROs still lack systematic characterization in tomato. Based on bioinformatics methods, SRO family genes were identified and characterized from cultivated tomatoes and several wild tomatoes. qRT-PCR was used to study the expression of SRO gene in cultivated tomatoes. Phylogenetic and evolutionary analyses showed that SRO genes in angiosperms share a common ancestor and that the number of SRO family members changed as plants diverged and evolved. Cultivated tomato had six SRO members, five of which still shared some degree of identity with the ancestral SRO genes. Genetic structure and physicochemical properties showed that tomato SRO genes were highly conserved with chromosomal distribution. They could be divided into three groups based on exon-intron structure, and cultivated tomato contained only two of these subclades. A number of hormonal, light and abiotic stress-responsive cis-regulatory elements were identified from the promoter of the tomato SRO gene, and they also interacted with a variety of stress-responsive proteins and microRNAs. RNA-seq analysis showed that SRO genes were widely expressed in different tissues and developmental stages of tomato, with significant tissue-specific features. Expression analysis also showed that SRO genes respond significantly to high temperature and salt stress and mediate the tomato hormone regulatory network. These results provide a theoretical basis for further investigation of the functional expression of tomato SRO genes and provide potential genetic resources for tomato resistance breeding.

Genome-Wide Analysis and Expression Profiling of the Phospholipase D Gene Family in Solanum tuberosum

Phospholipase D (PLD) is the most important phospholipid hydrolase in plants, which can hydrolyze phospholipids into phosphatidic acid (PA) and choline. When plants encounter low temperature, drought and high salt stress, phospholipase D and its products play an important role in regulating plant growth and development and coping with stress. In this study, 16 members of StPLD gene family were identified in potato genome, which were distributed in α, β, δ, and ζ subfamilies, and their expression patterns under salt, high temperature, drought, and ABA stress were detected by qRT-PCR method. Gene expression analysis showed that the expression of StPLD genes in potato was upregulated and downregulated to varying degrees under the four stresses, indicating that the PLD gene family is involved in the interaction of potato plant hormones and abiotic stress signals. Chromosome distribution showed that StPLD gene was unevenly distributed on 8 chromosomes, and only one pair of tandem repeat genes was found. All StPLD promoters contain hormone and stress-related cis-regulatory elements to respond to different stresses. Structural analysis showed that StPLD genes in the same subgroup had a similar exon–intron structure. Our study provides a valuable reference for further research of the function and structure of PLD gene.

Analysis of Expression and Bioinformatics of Trehalose-6-phosphate Phosphatases (TPP) Gene Family in Wheat Indicates a Role in Plant Development and Stress Response

Background: Trehalose-6-phosphate phosphatases genes (TPPs) are involved in the development and stress response of plants by regulating the biosynthesis of trehalose, though little is currently known about TPPs in common wheat (Triticum aestivum L.).Results: In this study, we performed a genome-wide identification of the TPP gene family in common wheat, and identified a total of 31 TPP genes. These were subdivided into six subfamilies based on the phylogenetic relationships and the conservation of protein in six monocot and eudicot plants. The majority of TPP genes were represented by 2-3 wheat homoalleles (named TaTPPX_ZA, TaTPPX_ZB, or TaTPPX_ZD), where Z is the location on the wheat chromosome of the gene number (X). We also analyzed the chromosomal location, exon-intron structure, orthologous genes, and protein motifs of the TaTPPs. The RNA-seq data was used to perform an expression analysis, which found 26 TaTPP genes to be differentially expressed based on spatial and temporal characteristics, indicating they have varied functions in the growth and development of wheat. Additionally, we assessed how the promoter regulatory elements were organized and used qRT-PCR in the leaves to observe how they were expressed following ABA, salt, low tempreture, and drought stress treatments. All of these genes exhibited differential expression against one or more stress treatments. Furthermore, overexpressing TaTPP11 in Arabidopsis results delayed plant development and enhanced drought tolerance, but not affect seed morphology. Conclusions: TaTPPs could serve important roles in the development and stress response in wheat. These results provide a basis for subsequent research into the function of TaTPPs.

Identification, Characterization, and Expression Profile Analysis of the mTERF Gene Family and Its Role in the Response to Abiotic Stress in Barley (Hordeum vulgare L.)

Plant mitochondrial transcription termination factor (mTERF) family regulates organellar gene expression (OGE) and is functionally characterized in diverse species. However, limited data are available about its functions in the agriculturally important cereal barley (Hordeum vulgare L.). In this study, we identified 60 mTERFs in the barley genome (HvmTERFs) through a comprehensive search against the most updated barley reference genome, Morex V2. Then, phylogenetic analysis categorized these genes into nine subfamilies, with approximately half of the HvmTERFs belonging to subfamily IX. Members within the same subfamily generally possessed conserved motif composition and exon-intron structure. Both segmental and tandem duplication contributed to the expansion of HvmTERFs, and the duplicated gene pairs were subjected to strong purifying selection. Expression analysis suggested that many HvmTERFs may play important roles in barley development (e.g., seedlings, leaves, and developing inflorescences) and abiotic stresses (e.g., cold, salt, and metal ion), and HvmTERF21 and HvmTERF23 were significant induced by various abiotic stresses and/or phytohormone treatment. Finally, the nucleotide diversity was decreased by only 4.5% for HvmTERFs during the process of barley domestication. Collectively, this is the first report to characterize HvmTERFs, which will not only provide important insights into further evolutionary studies but also contribute to a better understanding of the potential functions of HvmTERFs and ultimately will be useful in future gene functional studies.

Genome-Wide Analysis of the YABBY Transcription Factor Family in Rapeseed (Brassica napus L.)

The YABBY family of plant-specific transcription factors play important regulatory roles during the development of leaves and floral organs, but their functions in Brassica species are incompletely understood. Here, we identified 79 YABBY genes from Arabidopsis thaliana and five Brassica species (B. rapa, B. nigra, B. oleracea, B. juncea, and B. napus). A phylogenetic analysis of YABBY proteins separated them into five clusters (YAB1–YAB5) with representatives from all five Brassica species, suggesting a high degree of conservation and similar functions within each subfamily. We determined the gene structure, chromosomal location, and expression patterns of the 21 BnaYAB genes identified, revealing extensive duplication events and gene loss following polyploidization. Changes in exon–intron structure during evolution may have driven differentiation in expression patterns and functions, combined with purifying selection, as evidenced by Ka/Ks values below 1. Based on transcriptome sequencing data, we selected nine genes with high expression at the flowering stage. qRT-PCR analysis further indicated that most BnaYAB family members are tissue-specific and exhibit different expression patterns in various tissues and organs of B. napus. This preliminary study of the characteristics of the YABBY gene family in the Brassica napus genome provides theoretical support and reference for the later functional identification of the family genes.

Genome Wide Characterization & Phylogenetic Analysis of TNF Genes in Homo Sapiens

Tumor Necrosis Factor is very important inflammatory signaling unit that do an important role in immune system. It functions by attachment and stimulation of different receptor’s Cysteine Rich Domains (CDRs). A number of TNF receptors mediated factors have been identified having a major role in signal transduction pathways of TNF gene family. There are about 18 TNF homologues that are identified as a major cause of many disorders like cancer, Diabetes, AIDS and many other lethal inflammations. In this study the genome wide identification of TNF gene was done. Different tools and databases were used. Identification of conserved domains was done by using pfam and homology analysis showed that the TNF might be a member of TRAFs superfamily. Structural analysis of gene showed the number of introns and exons by a three-dimensional structure of TNF gene. The TNF gene family's exon-intron structure was found to be very similar in this study. The distribution of genes across chromosomes, on the other hand, was extremely varied. Collectively, the newly discovered genes can provide a wealth of information for manipulating the TNF genome to develop drugs and strategies to treat a variety of diseases.

On the relation of gene essentiality to intron structure: a computational and deep learning approach

Essential genes have been studied by copy number variants and deletions, both associated with introns. The premise of our work is that introns of essential genes have distinct characteristic properties. We provide support for this by training a deep learning model and demonstrating that introns alone can be used to classify essentiality. The model, limited to first introns, performs at an increased level, implicating first introns in essentiality. We identify unique properties of introns of essential genes, finding that their structure protects against deletion and intron-loss events, especially centered on the first intron. We show that GC density is increased in the first introns of essential genes, allowing for increased enhancer activity, protection against deletions, and improved splice site recognition. We find that first introns of essential genes are of remarkably smaller size than their nonessential counterparts, and to protect against common 3′ end deletion events, essential genes carry an increased number of (smaller) introns. To demonstrate the importance of the seven features we identified, we train a feature-based model using only these features and achieve high performance.