RAD gene family analysis in cotton provides some key genes for flowering and stress tolerance in upland cotton G. hirsutum

Background RADIALIS (RAD), belongs to the MYB gene family and regulates a variety of functions including floral dorsoventral asymmetry in Antirrhinum majus and development of fruit proteins in Solanum lycopersicum. RAD genes contain an SNF2_N superfamily domain. Here, we comprehensively identified 68 RAD genes from six different species including Arabidopsis and five species of cotton. Results Phylogenetic analysis classified RAD genes into five groups. Gene structure, protein motifs and conserved amino acid residues indicated that GhRAD genes were highly conserved during the evolutionary process. Chromosomal location information showed that GhRAD genes were distributed unevenly on different chromosomes. Collinearity and selection pressure analysis indicated RAD gene family expansion in G. hirsutum and G. barbadense with purifying selection pressure. Further, various growth and stress related promotor cis-acting elements were observed. Tissue specific expression level indicated that most GhRAD genes were highly expressed in roots and flowers (GhRAD2, GhRAD3, GhRAD4 and GhRAD11). Next, GhRAD genes were regulated by phytohormonal stresses (JA, BL and IAA). Moreover, Ghi-miRN1496, Ghi-miR1440, Ghi-miR2111b, Ghi-miR2950a, Ghi-miR390a, Ghi-miR390b and Ghi-miR7495 were the miRNAs targeting most of GhRAD genes. Conclusions Our study revealed that RAD genes are evolutionary conserved and might be involved in different developmental processes and hormonal stress response. Data presented in our study could be used as the basis for future studies of RAD genes in cotton.

qPCR Analysis Reveals Association of Differential Expression of SRR, NFKB1, and PDE4B Genes With Type 2 Diabetes Mellitus

BackgroundType 2 diabetes mellitus (T2DM) is a heterogeneous, metabolic, and chronic condition affecting vast numbers of the world’s population. The related variables and T2DM associations have not been fully understood due to their diverse nature. However, functional genomics can facilitate understanding of the disease. This information will be useful in drug design, advanced diagnostic, and prognostic markers.AimTo understand the genetic causes of T2DM, this study was designed to identify the differentially expressed genes (DEGs) of the disease.MethodsWe investigated 20 publicly available disease-specific cDNA datasets from Gene Expression Omnibus (GEO) containing several attributes including gene symbols and clone identifiers, GenBank accession numbers, and phenotypic feature coordinates. We analyzed an integrated system-level framework involving Gene Ontology (GO), protein motifs and co-expression analysis, pathway enrichment, and transcriptional factors to reveal the biological information of genes. A co-expression network was studied to highlight the genes that showed a coordinated expression pattern across a group of samples. The DEGs were validated by quantitative PCR (qPCR) to analyze the expression levels of case and control samples (50 each) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene.ResultsFrom the list of 50 DEGs, we ranked three T2DM-related genes (p < 0.05): SRR, NFKB1, and PDE4B. The enriched terms revealed a significant functional role in amino acid metabolism, signal transduction, transmembrane and intracellular transport, and other vital biological functions. DMBX1, TAL1, ZFP161, NFIC (66.7%), and NR1H4 (33.3%) are transcriptional factors associated with the regulatory mechanism. We found substantial enrichment of insulin signaling and other T2DM-related pathways, such as valine, leucine and isoleucine biosynthesis, serine and threonine metabolism, adipocytokine signaling pathway, P13K/Akt pathway, and Hedgehog signaling pathway. The expression profiles of these DEGs verified by qPCR showed a substantial level of twofold change (FC) expression (2−ΔΔCT) in the genes SRR (FC ≤ 0.12), NFKB1 (FC ≤ 1.09), and PDE4B (FC ≤ 0.9) compared to controls (FC ≥ 1.6). The downregulated expression of these genes is associated with pathophysiological development and metabolic disorders.ConclusionThis study would help to modulate the therapeutic strategies for T2DM and could speed up drug discovery outcomes.

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

Exploration of Autophagy Families in Legumes and Dissection of the ATG18 Family with a Special Focus on Phaseolus vulgaris

Macroautophagy/autophagy is a fundamental catabolic pathway that maintains cellular homeostasis in eukaryotic cells by forming double-membrane-bound vesicles named autophagosomes. The autophagy family genes remain largely unexplored except in some model organisms. Legumes are a large family of economically important crops, and knowledge of their important cellular processes is essential. Here, to first address the knowledge gaps, we identified 17 ATG families in Phaseolus vulgaris, Medicago truncatula and Glycine max based on Arabidopsis sequences and elucidated their phylogenetic relationships. Second, we dissected ATG18 in subfamilies from early plant lineages, chlorophytes to higher plants, legumes, which included a total of 27 photosynthetic organisms. Third, we focused on the ATG18 family in P. vulgaris to understand the protein structure and developed a 3D model for PvATG18b. Our results identified ATG homologs in the chosen legumes and differential expression data revealed the nitrate-responsive nature of ATG genes. A multidimensional scaling analysis of 280 protein sequences from 27 photosynthetic organisms classified ATG18 homologs into three subfamilies that were not based on the BCAS3 domain alone. The domain structure, protein motifs (FRRG) and the stable folding conformation structure of PvATG18b revealing the possible lipid-binding sites and transmembrane helices led us to propose PvATG18b as the functional homolog of AtATG18b. The findings of this study contribute to an in-depth understanding of the autophagy process in legumes and improve our knowledge of ATG18 subfamilies.

Investigation and Computational Analysis of the Sulfotransferase (SOT) Gene Family in Potato (Solanum tuberosum): Insights into Sulfur Adjustment for Proper Development and Stimuli Responses

Various kinds of primary metabolisms in plants are modulated through sulfate metabolism, and sulfotransferases (SOTs), which are engaged in sulfur metabolism, catalyze sulfonation reactions. In this study, a genome-wide approach was utilized for the recognition and characterization of SOT family genes in the significant nutritional crop potato (Solanum tuberosum L.). Twenty-nine putative StSOT genes were identified in the potato genome and were mapped onto the nine S. tuberosum chromosomes. The protein motifs structure revealed two highly conserved 5′-phosphosulfate-binding (5′ PSB) regions and a 3′-phosphate-binding (3′ PB) motif that are essential for sulfotransferase activities. The protein–protein interaction networks also revealed an interesting interaction between SOTs and other proteins, such as PRTase, APS-kinase, protein phosphatase, and APRs, involved in sulfur compound biosynthesis and the regulation of flavonoid and brassinosteroid metabolic processes. This suggests the importance of sulfotransferases for proper potato growth and development and stress responses. Notably, homology modeling of StSOT proteins and docking analysis of their ligand-binding sites revealed the presence of proline, glycine, serine, and lysine in their active sites. An expression essay of StSOT genes via potato RNA-Seq data suggested engagement of these gene family members in plants’ growth and extension and responses to various hormones and biotic or abiotic stimuli. Our predictions may be informative for the functional characterization of the SOT genes in potato and other nutritional crops.

Comprehensive Analysis of the SUV Gene Family in Allopolyploid Brassica napus and Its Diploid Ancestors

SUV (the Suppressor of variegation [Su(var)] homologs and related) gene family is a subgroup of the SET gene family. According to the SRA domain and WIYLD domain distributions, it can be divided into two categories, namely SUVH (the Suppressor of variegation [Su(var)] homologs) and SUVR (the Suppressor of variegation [Su(var)] related). In this study, 139 SUV genes were identified in allopolyploid Brassica napus and its diploid ancestors, and their evolutionary relationships, protein properties, gene structures, motif distributions, transposable elements, cis-acting elements and gene expression patterns were analyzed. Our results showed that the SUV gene family of B. napus was amplified during allopolyploidization, in which the segmental duplication and TRD played critical roles. After the separation of Brassica and Arabidopsis lineages, orthologous gene analysis showed that many SUV genes were lost during the evolutionary process in B. rapa, B. oleracea and B. napus. The analysis of the gene and protein structures and expression patterns of 30 orthologous gene pairs which may have evolutionary relationships showed that most of them were conserved in gene structures and protein motifs, but only four gene pairs had the same expression patterns.

Genome-wide identification and expression profiling of duplicated flavonoid 3'-hydroxylase gene family in Carthamus tinctorius L.

Flavonoid 3′-hydroxylase (F3’H) enzyme is essential in determining the flavonoids B-ring hydroxylation pattern. It is mainly implicated in the biosynthetic pathway of cyaniding-based anthocyanins, flavonols, and flavan-3-ols. However, the evolution and regulatory mechanism of these important flavonoid hydroxylases have not been systematically investigated in safflower (Carthamus tinctorius L.). In this study, we identified 22 duplicatedCtF3'H-encoding genes from safflower through genome-wide prediction and conservation analysis. Phylogenetic analysis revealed the pattern of conservation and divergence of CtF3'Hs encoding proteins and their homologs from different plant species. The distribution of conserved protein motifs and cis-regulatory units suggested several structural components that could be crucial in deciphering the final function of CtF3'H proteins. Furthermore, the results of RNA-seq and qRT-PCR assay in different flowering tissues suggested differential expression level of CtF3’H genes during flower development. Based on the unique homology of CtF3’H5 with flavonoid 3’ hydroxylases from other plant species, further validation of CtF3’H5 was carried out. The transient expression of CtF3’H5 in onion epidermal cells implied that the subcellular localization of the fusion construct containing CtF3’H5 and GFP was predominantly detected in the plasma membrane. Similarly, the prokaryotic expression and western blot hybridization of CtF3’H5 demonstrated the detection of a stable 50.3kD target protein. However, more efforts are needed to further extend the functional validation of CtF3’H5 in safflower. This study provides a fundamental gateway for future functional studies and understanding the genetic evolution of F3'Hs in plants.

Genome-Wide Identification of CBL-CIPK Gene Family in Honeysuckle (Lonicera japonica Thunb.) and Their Regulated Expression Under Salt Stress

In plants, calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating a family of plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). CBL-CIPK gene families and their interacting complexes are involved in regulating plant responses to various environmental stimuli. To gain insight into the functional divergence of CBL-CIPK genes in honeysuckle, a total of six LjCBL and 17 LjCIPK genes were identified. The phylogenetic analysis along with the gene structure analysis divided both CBL and CBL-interacting protein kinase genes into four subgroups and validated by the distribution of conserved protein motifs. The 3-D structure prediction of proteins shown that most LjCBLs shared the same Protein Data Bank hit 1uhnA and most LjCIPKs shared the 6c9Da. Analysis of cis-acting elements and gene ontology implied that both LjCBL and LjCIPK genes could be involved in hormone signal responsiveness and stress adaptation. Protein-protein interaction prediction suggested that LjCBL4 is hypothesized to interact with LjCIPK7/9/15/16 and SOS1/NHX1. Gene expression analysis in response to salinity stress revealed that LjCBL2/4, LjCIPK1/15/17 under all treatments gradually increased over time until peak expression at 72 h. These results demonstrated the conservation of salt overly sensitive pathway genes in honeysuckle and a model of Ca2+-LjCBL4/LjSOS3-LjCIPK16/LjSOS2 module-mediated salt stress signaling in honeysuckle is proposed. This study provides insight into the characteristics of the CBL-CIPK gene families involved in honeysuckle salt stress responses, which could serve as a foundation for gene transformation technology, to obtain highly salt-tolerant medicinal plants in the context of the global reduction of cultivated land.

Genome-Wide Identification and Functional Analysis of the Basic Helix-Loop-Helix (bHLH) Transcription Family Reveals Candidate PtFBH Genes Involved in the Flowering Process of Populus trichocarpa

As one of the largest TF families+ in plants, the basic helix-loop-helix (bHLH) family plays an important part in the growth and development of many plants. FLOWERING BHLH (FBH) encodes a bHLH-type transcriptional factor related to the flowering process. Poplar is a model woody plant as well as an important economic tree species with a small genome. However, the characteristics of the bHLHs and FBHs gene family in the newest version of Populustrichocarpa genome have not been analyzed yet. We identified 233 PtbHLHs and 10 PtFBHs in the newest version genome, and PtbHLHs were classified into 21 groups with FBH subfamily occupying one, supported by phylogenetic analysis, exon–intron patterns, and conserved protein motifs. These PtHLHs were distributed on 19 chromosomes unevenly and expressed in nucleus mainly. Gene duplication and synteny analysis have indicated that the PtbHLHs gene family has undergone strong purification selection during the evolution process. The cis-elements analysis has suggested that PtbHLHs may be related to the growth and development. Conserved residues of FBHs among Arabidopsis and poplar were also identified. Expression of 227 PtHLH genes (6 unmatched, 13 no expressed) showed diverse patterns in different tissues, implying their multiple functions. Protein–protein interaction network prediction and expression patterns in three states of the flowering process (Flowers-Dormant, Flowers-Expanding and Flowers-Expanded) suggested that some members of PtbHLH and PtFBH family may be involved in the flowering process. Our comprehensive and systematic analysis can provide some valuable clues and basic reference toward further investigations on physiological and molecular functions of PtbHLHs.

Genome-Wide Analysis of Major Facilitator Superfamily and Its Expression in Response of Poplar to Fusarium oxysporum

The major facilitator superfamily (MFS) is one of the largest known membrane transporter families. MFSs are involved in many essential functions, but studies on the MFS family in poplar have not yet been reported. Here, we identified 41 MFS genes from Populus trichocarpa (PtrMFSs). We built a phylogenetic tree, which clearly divided members of PtrMFS into six groups with specific gene structures and protein motifs/domains. The promoter regions contain various cis-acting elements involved in stress and hormone responsiveness. Genes derived from segmental duplication events are unevenly distributed in 17 poplar chromosomes. Collinearity analysis showed that PtrMFS genes are conserved and homologous to corresponding genes from four other species. Transcriptome data indicated that 40 poplar MFS genes were differentially expressed when treated with Fusarium oxysporum. Co-expression networks and gene function annotations of MFS genes showed that MFS genes tightly co-regulated and closely related in function of transmembrane transport. Taken together, we systematically analyzed structure and function of genes and proteins in the PtrMFS family. Evidence indicated that poplar MFS genes play key roles in plant development and response to a biological stressor.