Different fat ingredients and their fatty acids may influence the expression of genes responsible to encode enzymes and proteins linked to adipose tissue deposition, influencing beef quality. Therefore, the objective was to analyze the expression of PPARA, SREBF1 and genes involved in lipid metabolism in longissimus thoracis (LT) muscle of cattle fed diets containing soybean or cottonseed with or without vitamin E. Twenty-eight bulls were used in a completely randomized design using a 2 × 2 factorial arrangement. The inclusion of soybean and cottonseed in the diets provided 64.8 g kg−1 and 65.6 g kg−1 of ether extract, respectively, and vitamin E supplementation was 2500 IU per day. The PPARA expression was greater and SREBF1 expression was lower in the muscle of animals fed soybean (P < .01). Vitamin E reduced the expression of SREBF1 in animals fed cottonseed and increased its expression in animals fed soybean (P < .01). Expression of LPL and ACACA increased when bulls were fed with soybean (P < .01) and their expression was upregulated when animals received vitamin E (P < .03). Expression of SCD was higher when cottonseed and no vitamin E diet were used (P < .01). In conclusion, supplementation with vitamin E alters the expression of genes involved in lipid metabolism in LT muscle, indicating that α-tocopherol is a cell-signaling molecule.
Improving the efficiency of production to reduce the environmental footprints is pivotal to the sustainability of livestock systems. Despite the advances in cattle feed efficiency (FE) measurement and identification of potential mechanisms involved, much is still unclear regarding the genetic and biological basis of this trait. Nevertheless, lipid and carbohydrate metabolism have been outlined as important in determining efficient and inefficient animals. To address the role of genes partaking in these processes and previously involved with residual feed intake (RFI), we carried out a liver expression profile in Nelore steers (n = 83). Six target genes (FABP1, FADS2, PPP1R26, RGS2, SLC2A5, and UCP2) were measured by qPCR analysis. A general linear mixed model approach was applied to associate them with dry matter intake (DMI), body weight (BW), metabolic BW (MBW, kg), DMI as a percentage of BW (DMI%BW), and average daily gain (ADG, kg/d). Residual feed intake (RFI), feed conversion ratio (FCR), feed efficiency (FE), Kleiber index (KI), and relative growth rate (RGR) were also evaluated. Our results support that increased expression of FABP1 gene was associated with enhanced values for RFI and DMI. Likewise, higher expression level of SLC2A5 was related to higher KI and RGR. There was no phenotypic correlation between RFI and ADG, BW, and MBW. The positive correlations between FABP1 and SLC2A5, and between FABP1 and FADS2 gene expression suggest a putative co-regulation affecting feed efficiency phenotypes.
Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of vertebrates, involved in gonadal maturation and primarily regulated by the hypothalamic-pituitary-gonadal axis. The full length GnRH-like cDNA isolated from the cerebral ganglion of Haliotis discus hannai, encodes a deduced protein of 216 amino acids with a theoretical molecular mass of 23.36 kDa and an isoelectric point of 7.72. The GnRH transcript comprises a putative signal peptide, a GnRH dodecapeptide, a proteolytic processing site, and a GnRH associated peptide (GAP). Comparative structural analysis revealed that the cloned sequence shares relatively high homology with other molluscan species and a lower degree of amino acid identity with GnRH of piscine vertebrates. Phylogenetic comparison with other known GnRH genes revealed that the GnRH-like mRNA of H. discus hannai is most closely related to the marine gastropod, H. laevigata. Three-dimensional homology structure of H. discus hannai GnRH and GAP exhibited a helix-loop-helix structure. Quantitative PCR assay demonstrated wide expression of GnRH in all ganglia, among them cerebral ganglion exhibited the highest expression level. Significantly higher expression was observed in cerebral ganglion and gonadal tissues at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the GnRH expressing neurosecretory cells distributed throughout the cortex of the cerebral ganglion. These results suggest that GnRH synthesized from the cerebral ganglia may be involved in gonadal maturation and regulating the secretion of other reproductive hormones.
The larva of the Japanese buff-tip moth (Phalera flavescens) has a novel N-acetyllactosamine-binding lectin, termed Phalera flavescens agglutinin (PFA). It has been suggested that the PFA gene evolved from a lysozyme gene through the loss of lysozyme activity sites. In this study, we investigated whether the PFA gene is evolving through positive selection or by relaxation of functional constraint. For this purpose, we sequenced several PFA-orthologous genes from P. assimilis, P. takasagoensis, and P. bucephala, and compared synonymous and non-synonymous changes to examine the state of natural selection. Although a number of non-synonymous changes were observed on several branches of the gene tree, these effects may have been caused by relaxation of a functional constraint rather than a mechanism of positive selection. Therefore, it is reasonable to conclude that a lysozyme gene in the common ancestor of the Phalera (genus) species lost its lysozyme activity and accumulated mutations during the evolution of the species. Although the actual function of PFA in the moth species is as yet unknown, the gene probably obtained the N-acetyllactosamine-specific lectin activity, collaterally.
‘Candidatus Liberibacter asiaticus’ (Las) is one of the most destructive plant pathogens associated with citrus huanglongbing (HLB). Las is a Gram-negative, as yet uncultured, alpha-Proteobacterium and is transmitted by the Asian citrus psyllid, Diaphorina citri. A putative protein (designated as LasP235) was identified in the prophage region of the Las psy62 genome. LasP235 gene encodes a 123 amino-acid protein which is predicted to localize to the plant nucleus. Green fluorescence protein (GFP)-fused with LasP235 appeared to accumulate in Nicotiana benthamiana cell nuclei following Agrobacterium-mediated transient expression. To eliminate potential side effects of GFP protein, LasP235 alone was inserted into a binary vector for transforming Carrizo citrange (Citrus sinensis X Poncirus trifoliata). Transgenic Carrizo plants were obtained. LasP235 gene integration was confirmed by PCR and the levels of LasP235 expression were compared by RT-quantitative PCR (RT-qPCR). Some LasP235 expressing Carrizo plants displayed HLB-like symptoms, including leaf chlorosis and plant growth retardation. LasP235 gene expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. Furthermore, The expression of LasP235 was upregulated in chlorotic tissue compared to green tissue of Las-infected, blotchy mottled leaves of lemon and grapefruit. Transcriptome analysis revealed that metabolic pathways and biosynthesis of secondary metabolites were significantly altered in transgenic citrus expressing the LasP235 effector. Further investigation of LasP235, especially research focused on identifying its binding protein in citrus, may provide a way to block Las infection.
The entomopathogenic fungus Beauveria bassiana is used as a natural pest killer in many agricultural systems due to its power of overcoming the host immune system. This is partially due to its genome content that expresses key proteins involved in its virulence. In this study, we used Real-Time PCR technique to analyze the relative expression at 48 and 72 h post inoculation of the Hyd1, Hyd2, Bbchit1, Cdep1, Bbbeas, Bbbsls and Vlp4 genes implicated in the pathogenesis process of an indigenous strain (LTB) and a commercial strain (IND) of B. bassiana. This analysis revealed a higher induction of Bbbeas, Bbbsls and Vlp4 genes by the LTB strain when comparing to the IND strain and the calibrator. Furthermore, a more rapid repression of Hyd1 and Hyd2 gene was notable by LTB strain. No significant difference was recorded by the expression of the Cdep1 gene between the test samples and the control. Finally, a significant difference was recorded with the expression of Bbchit1 gene by the IND strain 72 h post-inoculation. In conclusion, these results suggest that B. bassiana strains have differential expression of virulence genes that could reflect adaptation to their geographical environment and could help classifying their entomopathogenic efficacy accordingly.
Fighting late blight, economically the most important of potato diseases, is greatly hampered by rapid changes in the populations of the pathogen Phytophthora infestans: new pathotypes turn up owing to pathogen evolution and migration and overcome potato varieties that were previously considered resistant. Early recognition of the changes in P. infestans populations would greatly assist potato protection against late blight. The pathogenicity of P. infestans depends on the (a)virulence genes (Avr genes), among which the best known are the genes encoding RXLR effectors. This is the first report on polymorphism of these genes when characterized by single-strand conformation polymorphism (SSCP) analysis. Highly reproducible SSCP patterns combine electrophoretic zones corresponding to the Avr alleles. Ten Avr genes were studied in two series of P. infestans samples: single cell lines obtained from the isolates collected in the potato genetic collection of the N.I. Vavilov Institute of Plant Genetic Resources (VIR), Pushkin, St. Petersburg, and the reference lines from the Western European and USA collections. The Avr3b, Avr4, and Avr8 genes were monomorphic, while in Avr1, Avr2, Avr3a, Avr9, Avr-blb1, Avr-blb2, and Avr-vnt1, we discerned two to five SSCP patterns, with their frequencies different in two series of P. infestans lines. Sequences of Avr alleles differed in synonymous and non-synonymous single nucleotide substitutions, with 93–100% identity between variants. When Avr polymorphisms were compared with the profiles of virulence factors recognized with the Mastenbroeck-Black differentials, the acceptable agreement between two independent indices was observed only for Avr1, Avr3a and Avr4.
Contagious ecthyma is a zoonotic disease, caused by the Orf virus (ORFV) affecting sheep, goats, and humans and is widely distributed across the world. In this study, we have investigated fifteen recent ORFV outbreaks in Black Bengal goats occurred in the Odisha state of India. The outbreaks were characterized by the high rates of morbidity (reaching up to 100%) without any mortality in the infected goats. Molecular and phylogenetic analysis of four ORFV genes such as ORFV011, ORFV020, ORFV059, and ORFV108 was performed, which revealed that current isolates were closely related to Assam, China, Bangalore, and Meghalaya isolates, respectively, with some unique mutations. Finally, to get an insight into the global ORFV evolution pattern, population genetics parameters such as nucleotide diversity, haplotype diversity, selection pressure, and neutrality among these genes were estimated by comparing the GenBank database. We observed low haplotype as well as nucleotide diversity and purifying selection implying ORFV evolution globally.
The ascomycete Colletotrichum falcatum is the causal agent of red rot of sugarcane, infecting stalks which are economically important for extraction of sugar. Since the pathogen is considered as the most destructive disease in many sugarcane growing countries it gained much attention to decipher its lifestyle. In this study, we have sequenced in planta transcriptome of C. falcatum under Illumina Hi-Seq platform and expanded our search towards finding the Small Secretory Proteins (SSPs) expressed during the host-pathogen interactions. Our previous reports from genome and transcriptome of C.falcatum data provided 768 and 884 SSPs were predicted respectively. The in planta secretory proteins were further mapped and localized with the help of bioinformatics pieplines such as TargetP and SignalP with the default parameters resulted in localizing 739 sequences to secretory pathway, 27 as mitochondrion and two contained chloroplast signal peptides. Further, the predicted secretory proteins were grouped into classical and non-classical proteins and these secreted proteins and 56 transmembrane helices were classified using GO, which revealed that signal peptides have a probable role in stabilizing fungal secretory proteins in the host system during pathogenesis. These small secreted proteins were further identified as crucial key pathogenic determinants from in planta transcriptome analysis. The repertoire of C. falcatum effectors prediction from in vitro and in planta transcriptome identified several putative genes which are involved in biotrophy-necrotrophy in functionally diverse patterns and this will facilitate further analysis of stage specific genes, fungal pathogenicity determinants and characterizing the expression of SSPs in in planta.
The relationship between the diversity of chemosensory proteins (CSPs) and their limited gene repertoire is of great interest. We have previously demonstrated that mRNA editing is responsible for this diversity. In this report, we describe hypotheses we have developed from phylogenetic analyses of CSP genes and RNA variants of the silkworm moth, Bombyx mori. Our analysis is based on previously published DNA/RNA/peptide sequencing data of the different variants from different tissues (Xuan et al., 2014, Xuan et al., 2016). We find and describe conventional A-to-I RNA editing, and C-to-U/U-to-C editing events in different CSP variants and pinpoint loci we believe encode the ADAR and ADAT enzymes and variants thereof that are responsible for these events. We also describe several non-conventional editing events including G-to-I and C-to-I, suggesting the existence of a multitude of mutation mechanisms in insects as found in microbes, viruses and plants. In particular, our phylogenetic analysis indicates that some mRNA sequences are more closely related to edited mRNA than to the gene sequence and shows evidence of tissue-specific DNA-dependent (Dd) or RNA-dependent (Rd) mutations depending on the gene. These descriptive findings in the silkworm are useful for molecular evolution of Dd-and Rd-RNA polymerases in relation with a specific gene family, as well as evolutionary biology and adaptation of RNA, cell, tissue or organism to new environment.
A maize gene coding for an E2F regulatory protein located in a quantitative trait locus (QTL) for Fusarium ear rot resistance was cloned and transgenically introduced into maize callus. Several of the transformants were more resistant to feeding by corn earworms and fall armyworms, and retarded growth of Fusarium proliferatum compared to GUS control transformants. More effective transformants contained higher levels of E2F product than GUS controls as indicated by antibody detection. Increased and decreased levels of regulated proteins were noted in E2F overexpressing compared to GUS expressing control transformants. These differentially produced proteins were previously reported to be interactors with the E2F protein, suggesting E2F is affecting the production of these proteins. Other proteins coded for by genes reported to interact with the relevant E2F protein and associated phenotypic effects that could be promoting resistance include those that would increase cell resistance to water stress, increase the presence of reactive oxygen species, and increase the density of indigestible components. Although the full complex of responsible proteins regulated by the E2F examined that promote resistance to insects and fungi remains to be determined, overproduction of the E2F in transgenic callus enhances resistance to some maize insect and fungal pests.
Rice tungro disease (RTD) is the most important viral disease of rice which limits its production and productivity in major rice growing regions of the world. The disease is caused by the combined action of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The RTBV is primarily responsible for symptom development while RTSV encodes proteins required for virus transmission. In this study, we developed an RNAi gene construct containing highly conserved partial sequences of coat protein 3 (CP3) gene of RTSV. The transgenic rice plants were developed in the background of japonica rice cultivar Taipei-309 through Agrobacterium mediated genetic transformation. The transgene showed stable inheritance as determined by PCR and Southern hybridization analyses. Two homozygous transgenic lines from the T4 generation were inoculated by virus through viruliferous insect vectors. These lines showed highly resistant phenotypes against the tungro disease which was further supported by studies of plant morphology and yield attributes. Also, the transgenic lines showed an inability to transmit the virus complex which could be due to suppression of RTSV proteins involved in virus transmission through insects. This study shows that CP3 of RTSV is a key target gene for development of RNAi mediated tungro disease resistance in rice.
The Japanese apricot (Prunus mume Sieb. et Zucc.) is a typical climacteric fruit which loses commercial value during processing when the surface color turns yellow. In our study, treatment with nordihydroguaiaretic acid (NDGA), an abscisic acid (ABA) synthesis inhibitor, delayed the increase of ethylene and decrease of firmness towards ripening, and retarded the degradation of chlorophyll and the accumulation of carotenoids including β-carotene and lycopene through the expression of PmCHL (coding for chlorophyllase), PmGGPPS (coding for geranylgeranyl diphosphate synthase), PmPDS (coding for phytoene desaturase), PmPSY (coding for phytoene synthases), and PmLCYB (coding for lycopene beta-cyclase). NDGA inhibited the ABA synthesis through PmNCED1 and these results show that ABA may affect the ripening of the Japanese apricot by influencing ethylene production and the expression of PmACS (coding for ACC synthase), PmACO (ACC oxidase) and the ethylene-responsive gene PmERF (ethylene-responsive transcription factor), and thus that ethylene and ABA may interact during fruit ripening in climacteric fruit.
Yellow stem borer (YSB), Scirpophaga incertulas, (Walker) is one the most destructive pests in rice causing severe yield loss. Of the many approaches for managing insect pests, RNA interference (RNAi) is a viable option which can be employed by targeting the key insect genes that are involved in host-pest interactions. In this study, Acetylcholinesterase (AChE), a key gene of YSB was targeted for silencing through a RNAi approach. Specifically designed dsRNA from the alpha/beta hydrolase fold of YSB AChE was labeled with 5’ FAM (Fluorescent dye) and used in this study. The efficacy of dsRNA was evaluated through a systematic insect bioassay. Larval growth and mortality were observed for a period of 15 days after the treatment through the cut stem assays. The reduced larval length and weight were observed in the dsRNA treated samples which were correlated with the low gene expression compared to the untreated controls. These results suggest that AChE is a potential target for RNAi approach in insects.
Most studies on plant-insect interactions focus on the aboveground parts of plants, but the knowledge regarding the belowground interactions is increasing. Soil pests are at least equally dangerous to plant health and elicit plant defense mechanisms as well. Wireworms (Coleoptera: Elateridae) are common polyphagous soil pests of various crops, including economically relevant crops such as maize and potatoes. Their management with pesticides is often not successful or sustainable, and more research on biological alternatives is required. We aim at providing an overview of biological control methods under development or commercially available. Little is known about the natural enemies of wireworms, and the available work is often limited to laboratory experiments. The interest for using using microorganisms as biocontrol agent is increasing, and entomopathogenic fungi, nematodes, and bacteria represent promising alternatives to pesticides. The review discusses the combination of attractive semiochemicals with biological agents to improve wireworm monitoring and control, as well as research advances on these fronts.
Flacherie is a widely prevalent disease in silkworms that causes huge economic losses to the sericulture industry. The Bombyx mori bidensovirus (BmBDV) is one of the causative agents of this disease. A major gene, nsd-2 (non-susceptibility to densovirus-2), a putative BmBDV receptor conferring resistance under recessive mutation condition has previously been identified. The natural deletion occurring in the nsd-2 gene has contributed to the evolution of BmBDV resistant silkworm breeds. However, nsd-2 has not yet been utilized as a marker or as a transgene for the development of BmBDV resistant breeds. In this study, we have demonstrated the use of the functional marker, nsd-2 for the development of absolute BmBDV resistant productive mulberry silkworm breeds. We screened 49 productive bivoltine breeds and identified 28 breeds carrying the nsd-2 resistant allele. Sixteen breeds having the resistant allele were identified in heterozygous condition at the nsd-2 locus, so these breeds could not be identified as resistant carrier when challenged with virus inoculation. Further, we have shown that selecting nsd-2 resistant allele in homozygous condition, at the nsd-2 locus is sufficient for the development of BmBDV resistant productive breeds like CSR6-R and CSR26-R. We have also validated the developed breeds for resistance to BmBDV through bioassays and found absence of viral genome at the molecular level thereby demonstrating complete resistance upon BmBDV infection, in the developed breeds.
Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait locus was cloned from an inbred with reported resistance to Fusarium proliferatum and Fusarium verticillioides ear rot. Transgenic expression of the gene in maize callus reduced colonization by these two Fusarium species and also Fusarium graminearum relative to a β-glucuronidase (GUS) transformant control. Some transformants were also more insect resistant. The more fungal resistant transformant lines produced higher levels of headspace ethanol which were significantly associated with antifungal activity, especially for F. verticillioides. Maize pyruvate decarboxylase appears to have a moiety capable of interacting with the geranyl geranyl transferase, suggesting ethanol production is enhanced due to more efficient transfer of pyruvate through the mitochondrial membrane. Other undetermined mechanisms may also be enhancing resistance of the transformants to the Fusarium fungus, however. This is the first report of the involvement of a geranyl geranyl transferase-like sequence in fungal resistance in plants, and represents a novel mechanism for producing higher yielding and better quality maize.
At altitudes >1500 m, measurements of mean pulmonary arterial pressures (mPAP; mmHg) are a measure of pulmonary hypertension (PH) in cattle. Genotypes of a G/A single nucleotide polymorphism (SNP; rs208684340) in the endothelial PAS domain protein 1 (EPAS1) gene were determined for mPAP categories (low, moderate, high) on Angus cattle. The A allele of this SNP was postulated to be associated with altitude-induced PH. The first objective was to estimate allele and genotypic frequencies in Angus cattle at high- (elevation 1850 to 2800 m) and low-altitude (elevation 91 to 1192 m) ranches (n = 118; random sample of 1275). Percent of cattle at these low elevation ranches with genotypes G/G, G/A, and A/A were 64.4, 33.9, and 1.7%, respectively, and a minor allele frequency (MAF; A allele) of 18.6%. Bulls, steers, and heifers (n = 691) from 4 high-altitude Angus herds in Colorado and Wyoming were genotyped. Percent of cattle with genotypes G/G, G/A, and A/A were 48.6, 34.3, and 17.1%, respectively, with a MAF of 34.2%. Average mPAP was 40.5 ± 8.8 mmHg. Low, moderate, and high-mPAP category MAF were similar (χ2 = 3.03; P = 0.22), with values of 35.4, 31.0, and 37.8%, respectively. Additionally we sought to evaluate the genotype-to-phenotype association of this SNP with mPAP phenotypes (n = 532) from the Colorado State University Beef Improvement Center (elevation 2150 m) Angus herd. Linear mixed model analysis suggested genotype was not a predictor (P = 0.61) of mPAP. Results do not suggest the A allele of SNP in EPAS1 is associated with high altitude-induced PH in yearling Angus cattle.
Identification of genes responsible for pest resistance in maize will assist with breeding attempts to reduce crop losses and hazards due to toxins produced by mold infecting ears. A gene coding for a defensin-like gene was cloned from an inbred reported to be resistant to Fusarium proliferatum and Fusarium verticillioides ear rot, based on its location in a QTL associated with resistance to those and other species of ear rot molds that produce mycotoxins. The gene was expressed transgenically in maize callus and the construct presence confirmed in transformants by PCR analysis and by detection with antibody made to a portion of the protein. Positive transformants were more resistant to corn earworms (Helicoverpa zea) and fall armyworms (Spodoptera fruigperda) as indicated by significantly lower weights compared to control callus expressing a β–glucuronidase (GUS) gene. Positive transformants also had significantly less visible growth of F. proliferatum and F. verticillioides, but not Fusarium graminearum, than controls. This indicates for the first time a defensin that is active against both insects and fungi, thereby allowing for more effective breeding for resistance to both major classes of pests attacking maize.
The Matang Mangrove Forest Reserve (MMFR) in Malaysia has been acknowledged as the best sustainably-managed mangrove forest in the world. The management employed a 30-year rotation cycle to enable harvest of mangrove timbers, followed by replanting and series of thinnings. Information on soil microbial diversity and its functional capability in managed mangrove is scarce. To this end, we analysed high-throughput metagenomic datasets from two sites at MMFR with distinct features: the Virgin Jungle Forest and the harvested Productive Zone. Taxonomic classification of sequencing reads using metagenomics RAST (MG-RAST) revealed distinct differences at phylum and class level for bacteria present in both samples. Productive zone, which consisted of managed forest blocks, exhibited total organic carbon content that was 18.7 times higher than that of Virgin Jungle Forest. The Productive Zone microbial community was overabundant in genes related to carbohydrate metabolism, especially enzymes involved in the degradation and utilization of polysaccharides from plant cell wall. Functional analysis focusing on carbohydrate degrading enzymes revealed an array of enzymes involved in hemicellulose, cellulose and pectin utilization enzymes in Matang mangrove metagenomes. Overall, differences in taxonomic composition and function profile of soil microorganisms were observed between Productive Zone and Virgin Jungle Forest. The results presented in this study have important implications in understanding managed mangrove microbial assemblage and provide important resources for the discovery of bacterial species and enzymes involved in litter processing.
