Search Results (6)

As an important branch of the lipoxygenase (LOX) metabolism pathway, hydroperoxide lyase (HPL) is involved in regulating plant development and defense responses. However, the upstream regulatory mechanism of HPL remains unclear in soybean. In the present study, by analyzing the upstream promoter region of the GmHPL gene, cis-elements such as MYB motifs, G-box motifs, ERE motifs and W-box motifs were predicted, which were related to the stress response. Yeast one-hybrid was employed and two transcription factors were identified, GmERF36 and GmILR3. The orthologs of ERF36 and ILR3 in Arabidopsis were involved in pathogen stress. A dual-luciferase reporter assay verified the yeast one-hybrid results and indicated that GmERF36 and GmILR3 suppressed the expression of the GmHPL protein. The qRT-PCR results indicated that GmHPL and GmERF36 initially displayed inverse expression patterns within 24 h after Colletotrichum truncatum treatment (GmERF36 was upregulated while GmHPL was downregulated); then, both of them were upregulated before decreasing. The results indicated that the response of GmHPL to pathogen stress partially depended on GmERF36. Our study gives rise to new insights into the upstream regulatory network of the GmHPL gene. Full article

Lipoxygenase (LOX) is a dioxygenase that contains non-heme iron and plays a crucial role in regulating plant growth and development, signal transduction, and responses to both biotic and abiotic stresses. In this study, we identified 24 CsLOXs from the pan-genome of 12 cucumber (Cucumis sativus L.) accessions, with most CsLOX proteins exhibiting amino acid variations. To elucidate their functions, we examined the phylogenetic relationships, gene structures, conserved domains, promoter cis-elements, and collinearity of the 24 CsLOXs from the newly updated genome version 4.0 of ‘Chinese Long 9930’. The results indicated that CsLOXs can be categorized into three subfamilies: 9-LOX, Type I 13-LOX, and Type II 13-LOX. Additionally, promoter analysis revealed that the promoters of CsLOXs contain various cis-elements related to stress and hormone responses. The expression of CsLOXs demonstrated tissue specificity, with each CsLOX expressed in at least one tissue, and six CsLOXs expressed across all tissues. Furthermore, in the transcriptome data of cucumber responses to heat, cold, powdery mildew (PM), downy mildew (DM), and gray mold (GM) stresses, eight, four, eight, eight, and four CsLOXs exhibited differential expression, respectively. Notably, CsLOX22 responded to heat, cold, DM, and GM stresses. Our results provided a reference for further exploring the functions of CsLOXs in cucumber. Full article

Genetic engineering technology offers opportunities to improve many important agronomic traits in crops, including insect-resistance. However, genetically modified (GM) exogenous proteins in edible tissues of transgenic crops has become an issue of intense public concern. To advance the application of GM techniques in maize, a Cre/loxP-based strategy was developed for manipulating the transgenes in green tissues while locking them in non-green tissues. In the strategy, the site-specific excision can be used to switch on or off the expression of transgenes at specific tissues. In this work, two basic transgenic maize, named KEY, carrying the Cre gene, and LOCK, containing the Vip3A gene with a blocked element, were obtained based on their separate fusion gene cassettes. The expression level and concentration of Vip3A were observed with a high specific accumulation in the green tissues (leaf and stem), and only a small amount was observed in the root and kernel tissues in the KEY × LOCK hybrids. The insect resistance of transgenic maize against two common lepidopteran pests, Ostrinia furnacalis and Spodoptera frugiperda, was assessed in the laboratory and field. The results indicate that the hybrids possessed high resistance levels against the two pests, with mortality rates above 73.6% and damage scales below 2.4 compared with the control group. Our results suggest that the Cre/loxP-mediated genetic engineering approach has a competitive advantage in GM maize. Overall, the findings from this study are significant for providing a feasible strategy for transgenes avoiding expression in edible parts and exploring novel techniques toward the biosafety of GM plants. Full article

Dongfudou 3 is a highly sought-after soybean variety due to its lack of beany flavor. To support molecular breeding efforts, we conducted a genomic survey using next-generation sequencing. We determined the genome size, complexity, and characteristics of Dongfudou 3. Furthermore, we constructed a chromosome-level draft genome and speculated on the molecular basis of protein deficiency in GmLOX1, GmLOX2, and GmLOX3. These findings set the stage for high-quality genome analysis using third-generation sequencing. The estimated genome size is approximately 1.07 Gb, with repetitive sequences accounting for 72.50%. The genome is homozygous and devoid of microbial contamination. The draft genome consists of 916.00 Mb anchored onto 20 chromosomes, with annotations of 46,446 genes and 77,391 transcripts, achieving Benchmarking Single-Copy Orthologue (BUSCO) completeness of 99.5% for genome completeness and 99.1% for annotation. Deletions and substitutions were identified in the three GmLox genes, and they also lack corresponding active proteins. Our proposed approach, involving k-mer analysis after filtering out organellar DNA sequences, is applicable to genome surveys of all plant species, allowing for accurate assessments of size and complexity. Moreover, the process of constructing chromosome-level draft genomes using closely related reference genomes offers cost-effective access to valuable information, maximizing data utilization. Full article

The lipoxygenase (LOX) cascade is a source of bioactive oxylipins that play a regulatory role in plants, animals, and fungi. Soybean (Glycine max (L.) Merr.) LOXs are the classical models for LOX research. Progress in genomics has uncovered a large diversity of GmLOX isoenzymes. Most of them await biochemical investigations. The catalytic properties of recombinant soybean LOX2 (GmLOX2) are described in the present work. The GmLOX2 gene has been cloned before, but only for nucleotide sequencing, while the recombinant protein was not prepared and studied. In the present work, the recombinant GmLOX2 behavior towards linoleic, α-linolenic, eicosatetraenoic (20:4), eicosapentaenoic (20:5), and hexadecatrienoic (16:3) acids was examined. Linoleic acid was a preferred substrate. Oxidation of linoleic acid afforded 94% optically pure (13S)-hydroperoxide and 6% racemic 9-hydroperoxide. GmLOX2 was less active on other substrates but possessed an even higher degree of regio- and stereospecificity. For example, it converted α-linolenic acid into (13S)-hydroperoxide at about 98% yield. GmLOX2 showed similar specificity towards other substrates, producing (15S)-hydroperoxides (with 20:4 and 20:5) or (11S)-hydroperoxide (with 16:3). Thus, the obtained data demonstrate that soybean GmLOX2 is a specific (13S)-LOX. Overall, the catalytic properties of GmLOX2 are quite similar to those of GmLOX1, but pH is optimum. Full article

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns. Full article