Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.
An expressed napin storage protein gene fromBrassica rapa, BcNA1, has been cloned and sequenced. The gene is a member of a family of four to seven napin genes inB. rapaand is highly expressed in developing seeds. An expression cassette containing the DNA flanking the napin coding region of BcNA1 has been engineered and demonstrated to function appropriately, as compared with the gene's endogenous expression, in transgenic rapeseed using the β-glucuronidase reporter gene. TheB. rapaBcNA1 gene and aB. napusnapin gene, gNa, share extremely high nucleotide homology not only throughout their coding regions, but over a DNA locus comprising 4.3 kb. We suggest the gNa gene was contributed by the originalB. rapaprogenitor of the amphidiploidB. napus.
We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryo-genesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5' and 3' untranslated and flanking regions fused with a beta-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.
Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1-9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.
Oleosins are small hydrophobic abundant proteins localized in the oil bodies of plant seeds. An oleosin gene from the monocotyledonous maize (Zea mays L.) was transferred into the dicotyledonous Brassica napus L. using Agrobacterium-mediated transformation. (4,5). Each oleosin molecule contains a relatively hydrophilic N-terminal domain of -48 amino acid residues, a central completely hydrophobic domain of -77 amino acid residues, and an amphipathic a-helical domain of -33 amino acid residues at or near the C terminus. Although each of the two maize oleosins contains these three structural domains, their amino acid and gene nucleotide sequences are significantly homologous only in the central hydrophobic domains. A subsequent study shows that the oleosin from dicotyledonous carrot somatic embryos also contains the above three distinct structural domains and, again, its amino acid sequence is highly similar to those of the two maize oleosins only in the central hydrophobic domain (6).Oil bodies are synthesized during seed maturation by the following proposed mechanism (1). Triacylglycerols are synthesized in the endoplasmic reticulum and sequestered between the two phospholipid layers of the membrane at a particular region. A budding vesicle of triacylglycerols surrounded by a layer of phospholipids is then formed, and the mature vesicle is released into the cytosol as an oil body. In maize, the oleosins are synthesized in the rough endoplasmic reticulum with no appreciable co-or post-translational processing (7). The location and exact nature of the signal in the oleosin molecule required for the transport of the newly synthesized protein from the endoplasmic reticulum to the oil body are unknown. It has been proposed that this intracellular transport signal resides in the central domain of the oleosin in view of its long stretch of 77 hydrophobic amino acid residues and its highly conserved amino acid sequences among different oleosins (4).We have transformed Brassica napus (a dicotyledon) with a maize (a monocotyledon) gene encoding an oleosin of 18 kDa (referred to as maize oleosin hereafter). In the transformants containing the maize oleosin gene under the control of its own 5' and 3' regulatory elements, mRNA and oleosin were not detectable. However, in the transformants containing the maize oleosin gene under the control of regulatory elements of a Brassica gene encoding napin (a major seed storage protein), mRNA and oleosin were produced abundantly. In these latter transformants, the expression of the maize oleosin gene was under temporal and tissue-specific controls similar to those for the napin gene. Also, the maize oleosin was exclusively localized in seed oil bodies, indicating that maize and Brassica share highly similar oleosin targeting mechanisms. Herein we report our findings. MATERIALS AND METHODSConstruction of Binary Vectors Containing the Maize Oleosin Gene. The scheme for the construction of two binary vectors containing the maize oleosin structural gene is shown in Fig. 1.A 4.0-ki...
We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryogenesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5'and 3' untranslated and flanking regions fused with a P-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.
A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.