Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

BackgroundIn industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze–thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain.ResultsElite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze–thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield.ConclusionsA multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze–thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.How to cite: Zhang Q, Jin Y, Fang Y, et al. Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high gravity bioethanol fermentation. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.06.003     

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid,Myzus persicae

BackgroundPiercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns.ResultsPinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids.ConclusionsThese findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.How to citeUmer N, Naqvi RZ, Rauf I, et al. Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.004.     

Analysis of gene expression profiles in response to Sporisorium reilianum f. sp. zeae in maize (Zea mays L.)

BackgroundHead smut of maize, which is caused by Sporisorium reilianum f. sp. zeae (Kühn), is a serious disease in maize. In order to reveal the molecular mechanism of the resistance to head smut in maize, a microarray containing ∼ 14,850 probes was used to monitor the gene expression profiles between a disease resistant near isogenic line (NIL) and a highly susceptible inbred line after S. reilianum was injected with an artificial inoculation method.ResultsLevels of expression for 3,532 genes accounting for 23.8% of the total probes changed after inoculation. Gene Ontology analysis revealed that the differentially expressed genes participated in physiological and biochemical pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that plant–pathogen interaction, natural killer cell mediated cytotoxicity and benzoxazinoid biosynthesis pathways play important roles in resistance to head smut. Three head smut resistance-related candidate genes, CLAVATA1, bassinosteroid insensitive 1-associated receptor kinase 1 and LOC100217307 with leucine-rich repeat (LRR) conserved domains were identified, each of which is in maize mapping bin 2.09, a region previously shown to include a major QTL for head smut resistance. Furthermore, LOC100217307 was validated by quantitative real-time (qRT)-PCR inferring that this gene may be involved in the resistance to head smut of maize.ConclusionsThis study provided valuable information for cloning, functional analysis and marker assisted breeding of head smut resistance genes.     

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

BackgroundCultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by the Δ12 fatty acid desaturase (FAD) encoded by AhFAD2A and AhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C → A:T) of AhFAD2A and an “A” insertion of AhFAD2B resulted in high-oleic acid phenotype. Detection of AhFAD2 mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detect AhFAD2 genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detection AhFAD2 genotype of large number of breeding materials.ResultsHere, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC₄F₂ seeds was aabb.ConclusionsDue to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable for determining AhFAD2 genotype than other methods.     

Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program

BackgroundCultivated peanut (Arachis hypogaea. L) represents one of the most important oil crops in the world. Although much effort has been expended to characterize microsatellites or Simple Sequence Repeats (SSRs) in peanut, the quantity and quality of the markers in breeding applications remain limited. Here, genome-wide SSR characterization and marker development were performed using the recently assembled genome of the cultivar Tifrunner.ResultsIn total, 512,900 microsatellites were identified from 2556.9-Mb genomic sequences. Based on the flanking sequences of the identified microsatellites, 7757 primer pairs (markers) were designed, and further evaluated in the assembled genomic sequences of the tetraploid Arachis cultivars, Tifrunner and Shitouqi, and the diploid ancestral species, A. duranensis and A. ipaensis. In silico PCR analysis showed that the SSR markers had high amplification efficiency and polymorphism in four Arachis genotypes. Notably, nearly 60% of these markers were single-locus SSRs in tetraploid Arachis species, indicating they are more specific in distinguishing the alleles of the A and B sub-genomes of peanut. In addition, two markers closely related with purple testa color and 27 markers near to FAD2 genes were identified, which could be used for breeding varieties with purple testa and high-oleic acid content, respectively. Moreover, the potential application of these SSR markers in tracking introgressions from Arachis wild relatives was discussed.ConclusionsThis study reported the development of genomic SSRs from assembled genomic sequences of the tetraploid Arachis Tifrunner, which will be useful for diversity analysis, genetic mapping and functional genomics studies in peanut.How to cite: Ma J, Zhao Y, Chen H, et al. Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.004.     

Identification and characterization of profilin gene family in rice

BackgroundProfilin proteins (PRFs) are small (12–15 kD) actin-binding protein, which play a significant role in cytoskeleton dynamics and plant development via regulating actin polymerization. Profilins have been well documented in Arabidopsis, Zea mays L. as well as Phaseolus vulgaris, however no such fully characterization of rice (Oryza sativa L.) profilin gene family has been reported thus far.ResultIn the present study, a comprehensive genome-wide analysis of rice PRF genes was completed and three members were identified. OsPRF1 and OsPRF2 shared 98.5% similarity (6 nucleotide divergence), but the deduced amino acid sequences of OsPRF1 and OsPRF2 are fully identical. In contrast, the OsPRF3 presents relatively lower similarity with OsPRF1 and OsPRF2. Phylogenetic analysis also support that OsPRF1 has a closer relationship with OsPRF2. Expression pattern analysis revealed the differential expression of OsPRFs in tissues of mature plant, which suggested the potential spatial functional specificity for rice profilin genes. Subcellular localization analysis revealed the OsPRFs were localized in cytoplasm and nucleus and all of them could bind actin monomers. Furthermore, abiotic stresses and hormones treatments assay indicated that the three OsPRF genes could be differentially regulated, suggesting that OsPRF genes might participate in different stress processes in rice.ConclusionsTaken together, our study provides a comprehensive analysis of the OsPRF gene family and will provide a basis for further studies on their roles in rice development and in response to abiotic stresses.How to cite: Zhang Y, Dong G, Wu L, et al. Identification and characterization of profilin gene family in rice. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.004.     

Effect of dietary nutrition on tail fat deposition and evaluation of tail-related genes in fat-tailed sheep

BackgroundThe effects of dietary nutrition on tail fat deposition and the correlation between production performance and the Hh signaling pathway and OXCT1 were investigated in fat-tailed sheep. Tan sheep were fed different nutritional diets and the variances in tail length, width, thickness and tail weight as well as the mRNA expression of fat-related genes (C/EBPα, FAS, LPL, and HSL) were determined in the tail fat of sheep at three different growth stages based on their body weight. Furthermore, the correlations between tail phenotypes and the Hedgehog (Hh) signaling pathway components (IHH, PTCH1, SMO, and GLI1) and OXCT1 were investigated.ResultsC/EBPα, FAS, LPL, and HSL were expressed with differences in tail fat of sheep fed different nutritional diets at three different growth stages. The results of the two-way ANOVA showed the significant effect of nutrition, stage, and interaction on gene expression, except the between C/EBPα and growth stage. C/EBPα, FAS, and LPL were considerably correlated with the tail phenotypes. Furthermore, the results of the correlation analysis demonstrated a close relationship between the tail phenotypes and Hh signaling pathway and OXCT1.ConclusionsThe present study demonstrated the gene-level role of dietary nutrition in promoting tail fat deposition and related tail fat-related genes. It provides a molecular basis by which nutritional balance and tail fat formation can be investigated and additional genes can be identified. The findings of the present study may help improve the production efficiency of fat-tailed sheep and identify crucial genes associated with tail fat deposition.How to cite: Zeng J, Zhou S, Yang Y, et al. Effect of dietary nutrition on tail fat deposition and evaluation of tail-related genes in fat-tailed sheep. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.05.004.     

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BackgroundMaize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop.ResultsIn this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression–an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments.ConclusionsThis paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.How to cite: Arévalo-Gallegos S, Varela-Rodríguez H, Lugo-Aguilar H, et al. Transient expression of a green fluorescent protein in tobacco and maize chloroplast. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.008