Identifying potential RNAi targets in aphid species (Macrosiphum rosae, Rhopalosiphum maidis, Sitobion avenae, Toxoptera aurantii) in sub-tropical region with extreme summer

Abstract RNA interference is a useful and efficient tool that had been used to incorporate tolerance against different stresses. Five unigene sequences were selected from exotic grain aphid that were reported to be an ideal RNAi targets. Aphids (Macrosiphum rosae, Rhopalosiphum maidis, Sitobion avenae, Toxoptera aurantii) were reared on Rosa indica, Zea mays, Triticum aestivum, Chrysanthemum hibiscus, Solanum melongena, Abelmoschus esculentus. PCR of unigene 28469 and 21789 was positive for all aphids and there is no ortholog found for them so unique and effective to be used in RNAi technology. Cytochrome c oxidase was found to be positive for M. rosae, S. avenae, T. aurantii and negative for R. maidis. Zinc finger protein was found to be positive for R. maidis, S. avenae and negative for M. rosae, and T. aurantii. Cuticular proteins was found to be positive for S. avenae and M. rosae and negative for R. maidis and T. aurantii. Genes identified in the aphids are defense and as important structural genes and their suppression with RNAi technology will be important target to have insect resistant crops. From these gene sequences cytochrome c oxidase is reported as bar coding gene and can be used in future for interspecific genetic variation in these aphids. Results revealed the high expression of these sequences in local aphid species and can be used as RNAi target for them. That can be used in future or applications as pest management, monitoring and plant quarantine

The industrial applicability of purified cellulase complex indigenously produced by Trichoderma viride through solid-state bio-processing of agro-industrial and municipal paper wastes

An indigenous strain of Trichoderma viride produced high titers of cellulase complex in solid-state bio-processing of agro-industrial orange peel waste, which was used as the growth-supporting substrate. When the conditions of the SSF medium containing 15 g orange peel (50% w/w moisture) inoculated with 5 mL of inoculum were optimal, the maximum productions of endoglucanase (655 ± 5.5 U/mL), exoglucanase (412 ± 4.3 U/mL), and β-glucosidase (515 ± 3.7 U/mL) were recorded after 4 days of incubation at pH 5 and 35 °C. The enzyme with maximum activity (endoglucanase) was purified by ammonium sulfate fractionation and Sephadex G-100 column gel filtration chromatographic technique. Endoglucanase was 5.5-fold purified with specific activity of 498 U/mg in comparison to the crude enzyme. The enzyme was shown to have a molecular weight of 58 kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE). The shelf life profile revealed that the enzyme could be stored at room temperature (30 °C) for up to 45 days without losing much of its activity

Genetic transformation of sugarcane variety HSF-240 with marker gene GUS

In the current research an efficient transformation system for sugarcane was established. Shoot tip of variety HSF-240, excised from a six months old field grown plants were used as explant. For transformation, Agrobacterium tumefaciens strain EHA101 with vector pIG121 Hm, harboring GUS, HPTII and NPTII genes were used. HPTII is a hygromycin resistant while NPTII is a kanamycin resistant gene. Effects of Acetosyringone, duration of co-cultivation and pre-selection, concentration of cefotaxime and hygromycin in medium on transformation efficiency were studied. High transformation efficiency and 60% GUS expression was observed when 50 μM acetosyringone was added in the co-cultivation medium. Among different durations of co-cultivation, 48 h produced high (40%) transient GUS positives with an absolute control of bacterial growth. For pre-selection, seven days gave a high transformation efficiency of 10%. Cefotaxime concentration of 1000 mg/L proved optimal for pre-selection of the explants with efficient control of bacterial growth. A high regeneration (31%; P < 0.01) of the transformants was observed at 50 mg/L hygromycin. Presence of GUS gene was confirmed by PCR analysis and only the transgenic plants contained the 430 bp fragment of GUS gene. The new protocol developed in this study could be used for the efficient transformation of sugarcane with desired gene to produce insect/pest resistant, drought tolerant and high yielding sugarcane varieties in future

Heterologous expression and enhanced production of β-1,4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-β-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-β-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-d-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced β-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the β-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 β-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 β-glucanases from B. halodurans.How to cite: Zeeshan N, Naz S, Naz S et al. Heterologous expression and enhanced production of β-1,4-glucanase of Bacillus halodurans C-125 in E. coli. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.05.001. Keywords: Bacillus halodurans, Cellulases, Cellulose hydrolysis, Degradation of cellulose, Endo-1, 4-β-glucanases, Expression analysis, Heterologous expression, In silico protein characterization, IPTG, pET expression system, Plant cell wal

Phenylalanine Ammonia-Lyase (PAL) Genes Family in Wheat (Triticum aestivum L.): Genome-Wide Characterization and Expression Profiling

Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway and plays a vital role in adoption, growth, and development in plants but in wheat its characterization is still not very clear. Here, we report a genome-wide identification of TaPAL genes and analysis of their transcriptional expression, duplication, and phylogeny in wheat. A total of 37 TaPAL genes that cluster into three subfamilies have been identified based on phylogenetic analysis. These TaPAL genes are distributed on 1A, 1B, 1D, 2A, 2B, 2D, 4A, 5B, 6A, 6B, and 6D chromosomes. Gene structure, conserved domain analysis, and investigation of cis-regulatory elements were systematically carried out. Chromosomal rearrangements and gene loss were observed by evolutionary analysis of the orthologs among Triticum urartu, Aegilops tauschii, and Triticum aestivum during the origin of bread wheat. Gene ontology analysis revealed that PAL genes play a role in plant growth. We also identified 27 putative miRNAs targeting 37 TaPAL genes. The high expression level of PAL genes was detected in roots of drought-tolerant genotypes compared to drought-sensitive genotypes. However, very low expressions of TaPAL10, TaPAL30, TaPAL32, TaPAL3, and TaPAL28 were recorded in all wheat genotypes. Arogenate dehydratase interacts with TaPAL29 and has higher expression in roots. The analysis of all identified genes in RNA-seq data showed that they are expressed in roots and shoots under normal and abiotic stress. Our study offers valuable data on the functioning of PAL genes in wheat

GC–MS metabolomics profile of methanol extract of Acacia modesta gum and gum-assisted fabrication and characterization of gold nanoparticles through green synthesis approach

Hereunder, for the first time, we reported phytocompounds in the methanolic extract of Acacia modesta (AM) gum through Gas chromatography-mass spectrometry (GS-MS). Further, the AM gum aqueous solution was used for gold nanoparticles (AuNPs) synthesis through a simple, swift, eco-friendly, and less costly green synthesis approach.  A total of 108 phytocompounds (63 with nonpolar, 45 with polar column) were identified in the gum extract, which includes fatty acids, alcohols, sterols, aldehyde/ketones, furans, aromatic compounds, esters, phenols, terpenes, sugar derivatives, alkaloids, and flavones. From three used concentrations (5, 10, and 15 mg/mL) of the AM gum aqueous solution, 15 mg/mL gum solution resulted in more successful AuNPs synthesis with a smaller size, which was visualized by a rusty red color appearance. UV-Visible absorption spectroscopy revealed the characteristic surface plasmon resonance (SPR) of AuNPs in aqueous solution at 540 nm. Dynamic light scattering (DLS) measurement of NPs solution revealed a hydrodynamic diameter of 162 ± 02 nm with the highest gum concentration where core AuNPs diameter was 22 ± 03 nm, recorded by Transmission electron microscopy. Zeta potential revealed fair stability of AuNPs that was not decreased with time. Catalytic activity experiments revealed that AM gum based AuNPs can increase the rate of the reduction of methylene blue 10 times in comparison with AM gum extract alone. Results from this study showed that a diverse array of phytocompounds in AM gum can successfully reduce gold ions into gold nanoparticles, which can be used further in different pharmaceutical and industrial applications. </p

Adverse events following immunization with typhoid conjugate vaccine in an outbreak setting in Hyderabad, Pakistan

Pakistan is facing the world\u27s largest outbreak of extensively drug-resistant (XDR) Typhoid. Vaccination campaign for children aged 6 months to 10 years old with Typhoid Conjugate Vaccine (Typbar-TCV®) was conducted in high-risk areas of Hyderabad during 2018. About 207,000 children were vaccinated. Here we report the adverse events following immunization (AEFI) during the campaign. The campaign was carried out using outreach and fixed centre strategy. Community mobilizers visited each household to perform line listing and mobilize parents with age-eligible children. Children were observed for 30 min post-vaccination. Two-pronged strategy was used for ascertainment of AEFI. A 24/7 hotline number was provided to all parents/caretakers (n = 199,861) to report AEFI during 14 days following immunization. An age-stratified (n = 7139 children) were actively followed at days 7 and 14 for the ascertainment of AEFI. All AEFI were examined by three trained medical officers. A structured questionnaire using Brighton collaboration criteria with level 3 diagnostic certainty was used for the recording of AEFI. Data were analysed using Microsoft Excel Office 365. Overall, 499 AEFI (433 in the subset actively followed and 66 self-reported through hotline) were observed. The rate of AEFI was significantly higher among very young children (age group 6 to 12 months) as compared to 2 to 3 years old children (0.54% vs. 0.33% respectively; p-value \u3c 0.001). Fever was the most common AEFI self-reported through the hotline (38/199,861 = 0.02%) and among the subset followed actively for 14 days (206/7139 = 2.89%). Fever was followed by local reactogenicity 10/199,861(0.01%), and 134/7139 (1.88%) through self-reported hotline and active follow-up, respectively. No serious AEFI was observed. Administration of a single dose of Typbar-TCV among children aged 6 months to 10 years old during an outbreak setting of Hyderabad Pakistan was safe