BackgroundStevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking.ResultsHere, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42–54% and 67–88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants.ConclusionThis study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1600-2) contains supplementary material, which is available to authorized users.
Hybridization between plant species can induce speciation as well as phenotypic novelty and heterosis. Hybrids also can show genome rearrangements and gene expression changes compared with their parents. Here we determined the allelic variation in gene expression in Populus trichocarpa 3 Populus deltoides F 1 hybrids. Among 30 genes analyzed in four independently formed hybrids, 17 showed .1.5-fold expression biases for one of the two alleles, and there was monoallelic expression of one gene. Expression ratios of the alleles differed between leaves and stems for 10 genes. The results suggest differential regulation of the two parental alleles in the hybrids. To determine if the allelic expression biases were caused by hybridization we compared the ratios of species-specific transcripts between an F 1 hybrid and its parents. Thirteen of 19 genes showed allelic expression ratios in the hybrid that were significantly different from the ratios of the parental species. The P. deltoides allele of one gene was silenced in the hybrid. Modes of gene regulation were inferred from the hybrid-parent comparisons. Cis-regulation was inferred for 6 genes, trans-regulation for 1 gene, and combined cis-and trans-regulation for 9 genes. The results from this study indicate that hybridization between plant species can have extensive effects on allelic expression patterns, some of which might lead to phenotypic changes.
The transfer of functional mitochondrial genes to the nucleus is an ongoing process during plant evolution that has made a major impact on cytonuclear interactions and mitochondrial genome evolution. Analysis of evolutionarily recent transfers in plants provides insights into the evolutionary dynamics of the process and how transferred genes become functional in the nucleus. Here, we report 42 new transferred genes in various angiosperms, including 9 separate transfers of the succinate dehydrogenase gene sdh3. We performed comparative analyses of gene structures and sequence evolution of 77 genes transferred to the nucleus in various angiosperms, including multiple transfers of 10 genes in different lineages. Many genes contain mitochondrial targeting presequences, and potentially 5' cis-regulatory elements, that were acquired from pre-existing nuclear genes for mitochondrial proteins to create chimeric gene structures. In eight separate cases, the presequence was acquired from either the hsp70 chaperonin gene or the hsp22 chaperonin gene. The most common location of introns is in the presequence, and the least common is in the region transferred from the mitochondrion. Several genes have an intron between the presequence and the core region, or an intron in the 5'UTR (untranslated region) or 3'UTR, suggesting presequence and/or regulatory element acquisition by exon shuffling. Both synonymous and nonsynonymous substitution rates have increased considerably in the transferred genes compared with their mitochondrial counterparts, and the degree of rate acceleration varies by gene, species, and evolutionary timing of transfer. Pairwise and branchwise K(a)/K(s) analysis identified four genes with evidence for positive selection, but positive selection is generally uncommon in transferred genes. This study provides a detailed portrayal of structural and sequence evolution in mitochondrial genes transferred to the nucleus, revealing the frequency of different mechanisms for how presequences and introns are acquired and showing how the sequences of transferred genes evolve after movement between cellular genomes.
To analyse the inheritance of fruit ring rot (FRR) resistance and to screen for microsatellite markers linked to resistance⁄susceptibility, 875 apple hybrid seedlings (Malus domestica, Jonathan · Golden Delicious) were inoculated with five isolates of Botryosphaeria dothidea in 2 years (2008 and 2009). The results indicated that incidence and non-incidence were qualitatively segregated, and incidence was dominant to non-incidence. The variation in susceptibility within this population was attributed to the segregation of three major genes. For the phenotype of incidence, the severity of lesion development was a quantitative trait. From 230 published microsatellite primer pairs, six markers were identified that were linked to the susceptibility to FRR. located in LG12 and LG2, respectively, were linked to susceptibility to the pathogen isolate Mx1, and their map distances to the susceptibility loci were 8.2 and 5.1 centimorgan (cM), respectively. CH01e01-120 and CH02c02b-100, which were linked to susceptibility to Ls1, were located in LG14 and LG4, and the map distances to the susceptibility loci were 16.9 and 8.4 cM, respectively. CH05d11-150 and CH03a03-230, linked to susceptibility to Lw048, were located in LG12 and LG14; for both of them, the map distance was 13.4 cM.
Plant peroxidases (POXs) are one of the most important redox enzymes in the defense responses. However, the large number of different plant POX genes makes it necessary to carefully confirm the function of each paralogous POX gene in specific tissues and disease interactions. Fusarium wilt is a devastating disease of common bean caused by Fusarium oxysporum f. sp. phaseoli. In this study, we evaluated a peroxidase gene, PvPOX1, from a resistant common bean genotype, CAAS260205 and provided direct evidence for PvPOX1's role in resistance by transforming the resistant allele into a susceptible common bean genotype, BRB130, via hairy root transformation using Agrobacterium rhizogenes. Analysis of PvPOX1 gene over-expressing hairy roots showed it increased resistance to Fusarium wilt both in the roots and the rest of transgenic plants. Meanwhile, the PvPOX1 expressive level, the peroxidase activity and hydrogen peroxide (HO) accumulation were also enhanced in the interaction. The result showed that the PvPOX1 gene played an essential role in Fusarium wilt resistance through the occurrence of reactive oxygen species (ROS) induced hypersensitive response. Therefore, PvPOX1 expression was proven to be a valuable gene for further analysis which can strengthen host defense response against Fusarium wilt through a ROS activated resistance mechanism.
Reversion from triploids to diploids or heteroploid mosaics may make the revertants recover reproductive ability and lose their aquacultural advantages. Meiotic chromosomes in triploids and mosaics of Crassostrea gigas and Crassostrea ariakensis were studied. Pachytene spermatocytes showed incomplete trivalent formation, varying 64–72% in C. gigas and 62–69% in C. ariakensis. At metaphase I, trivalents, bivalents and univalents occurred in various combinations. Trivalents occurred in all spermatocytes, ranged 6–10 per spermatocyte and averaged 8.53–8.97 in C. gigas and 8.49–8.95 in C. ariakensis. Univalents and bivalents appeared in 59.2–72.1% of the spermatocytes in C. gigas, and ranged 0.77–1.39 and 1.01–1.51 per spermatocyte respectively. In C. ariakensis, they occurred in 57.5–81% of the spermatocytes, and ranged 0.9–1.32 and 1.03–1.35 respectively. The most common trivalent was formed by a bivalent with the third chromosome attaching to its side to form ‘long‐tail‐cross’ or ‘t’ or ‘frying‐pan’‐shaped configurations that constituted 65.7% and 59.9% of the trivalents in C. gigas and C. ariakensis respectively. Other kinds of trivalent associations included tandem chains (14.7%, 16.5%), closed circles or triangles (5.3%, 9.8%), convergent ‘T’ or ‘Y’ (5.8%, 7.4%) and unclassified configurations (8.5%, 6.4%). Diploid spermatocytes were not observed from mosaics, although they showed considerable proportions of ‘diploid’ cells in their gills.
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