A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants

Dalal, Jyoti; Yalamanchili, Roopa; Hovary, Christophe La; Ji, Mikyoung; Rodriguez-Welsh, Maria; Aslett, Denise; Ganapathy, Sowmya; Grunden, Amy M.; Sederoff, Heike; Qu, Rongda

doi:10.1016/j.plasmid.2015.06.003

Cited by 23 publications

(19 citation statements)

References 34 publications

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“…The Arabidopsis RuBisCO small subunit (RBCS) transit peptide [ 73 ] was selected for chloroplast targeting of GlcD, GlcF and TSR, while the biotin carboxyl carrier protein (BCCP) transit peptide [ 73 ] was chosen for chloroplast targeting of GlcE and GCL. The three individual gene constructs for GDH were cloned into the multiple cloning site of a modified pCAMBIA2300 binary vector (CAMBIA Institute http://www.cambia.org/ ), in which the NPTII gene for kanamycin-resistance selection in plants was replaced by the mCherry coding sequence from the plasmid ER-RB [ 74 , 75 ]. The resulting vector is called DEF2 (Fig.…”

Section: Methodsmentioning

confidence: 99%

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa

Dalal

Lopez

Vasani

et al. 2015

Biotechnol Biofuels

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106

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BackgroundCamelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits.ResultsExpressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50–57 %, while expression of full bypass increased seed yield by 57–73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions.ConclusionsThe photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0357-1) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa

Dalal

Lopez

Vasani

et al. 2015

Biotechnol Biofuels

Self Cite

106

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“…Synthetic Biology is influencing Plant Biotechnology primarily with the adoption of new cloning methods, now renamed as DNA assembly methods. A panoply of new assembly strategies have been developed based either on site-specific recombination (1), PCR-overlap (2,3) or Type IIS enzymes (4–6), which bring the efficiency required to facilitate complex multigene engineering. Type IIS systems based on the original Goldengate strategy (7) are particularly interesting in the context of Synthetic Biology, as they open the way for the definition of assembly standards that, if widely adopted, will facilitate the exchange of DNA parts.…”

Section: Introductionmentioning

confidence: 99%

GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

et al. 2017

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Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.

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“…To construct the N‐terminal KmR–GFP–PIPL I‐ Sce I insertion cassette, the KmR gene amplified (primers BamSceKm5 and Km3Spe; Table S1) as the Bam HI−I‐ Sce I/ Spe I fragment from pGEM‐KmR‐araC‐ccdB and the GFP–PIPL coding region amplified without a stop codon as Spe I−I‐ Sce I/ Kpn I fragment from pBSK–GFP–PIPL (primers GFPPIPL5SpeSce and GFPPIPL3Kpn) were cloned into Bam HI− Kpn I sites of pBSKII yielding pNKmR–GFP–PIPL. The Spe I− Kpn I GFP–PIPL fragment of the latter vector was replaced with the mCherry coding region amplified without stop codon as Spe I−I‐ Sce I/ Kpn I fragment from pPC‐GW–mCherry (Dalal et al ., ; primers mCher5SpeISceI and mCher3KpnI) to create the pN‐KmR–mCherry cassette plasmid. The Not I− Spe I fragments of KmR genes in the GFP–PIPL and mCherry cassettes were replaced by a Not I− Spe I fragment of the SpR gene amplified from pER8 (primers NotBamHI‐SceSpF and SpectR3) to construct the cassette plasmids pN‐SpR–GFP–PIPL and N‐SpR–mCherry.…”

Section: Methodsmentioning

confidence: 99%

Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

Ghosh

Stolze

et al. 2019

The Plant Journal

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To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His 18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

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A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants

Cited by 23 publications

References 34 publications

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa

GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

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