Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.

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Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors

Functional Plant Biology ◽

10.1071/pp00129 ◽

2001 ◽

Vol 28 (3) ◽

pp. 241 ◽

Cited By ~ 8

Author(s):

Hui-Juan Lu ◽

Xue-Rong Zhou ◽

Zhu-Xun Gong ◽

Narayana M. Upadhyaya

Keyword(s):

Selectable Marker ◽

Binary Vector ◽

Marker Gene ◽

Marker Genes ◽

Binary Vectors ◽

Selectable Marker Genes ◽

Right Border ◽

Marker Free ◽

Dna Vectors ◽

Transgenic Progeny

Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce selectable marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a selectable marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two selectable marker genes and successfully obtained progeny plants in which the second selectable marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.

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Regeneration and Transformation via Agrobacterium tumefaciens of Echinacea purpurea L.

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v20i2.6888 ◽

1970 ◽

Vol 20 (2) ◽

pp. 101-111 ◽

Cited By ~ 1

Author(s):

Moemen Hanafy ◽

Usama I. Aly ◽

Mohamed A. Matter

Keyword(s):

Selectable Marker ◽

Binary Vector ◽

Stem Elongation ◽

Leaf Explants ◽

Echinacea Purpurea ◽

Plant Genome ◽

Bar Gene ◽

Herbicide Resistant ◽

Glufosinate Ammonium ◽

Chitinase Genes

Leaf explants of Echinacea purpurea L. taken from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA105, carrying a binary vector conferring herbicide resistant bar gene and fungal resistant chitinase gene. Glufosinate ammonium-resistant shoots were regenerated on a medium containing BAP and NAA at a concentration of 4.88 and 0.053 µM, respectively. A subsequent transfer of shoots to medium containing BAP was necessary for stem elongation and leaf development. Transgenic Echinacea plants carrying bar and chitinase genes were selected for their resistance to glufosinate ammonium herbicide. Molecular analysis using PCR confirmed the integration of the transgenes into plant genome. This is the first report on genetic transformation of Echinacea plant using bar gene as a selectable marker. Key words: Echinacea purpurea; Regeneration; A. tumefaciens; Transformation D.O.I. 10.3329/ptcb.v20i2.6888 Plant Tissue Cult. & Biotech. 20(2): 101-111, 2010 (December)

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An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2520-2525.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2520-2525 ◽

Cited By ~ 68

Author(s):

Alexandra E. Bloor ◽

Rocky M. Cranenburgh

Keyword(s):

Antibiotic Resistance ◽

Resistance Gene ◽

Selectable Marker ◽

Antibiotic Resistance Gene ◽

Marker Gene ◽

Stable Gene ◽

Selectable Marker Gene ◽

Site Specific ◽

Target Locus ◽

Bacterial Chromosomes

ABSTRACT A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.

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605 Agrobacterium-mediated Cranberry (Vaccinium macrocarpon) Transformation

HortScience ◽

10.21273/hortsci.35.3.501c ◽

2000 ◽

Vol 35 (3) ◽

pp. 501C-501

Author(s):

Luping Qu ◽

James Polashock ◽

Nicholi Vorsa

Keyword(s):

Transformation Efficiency ◽

Selectable Marker ◽

Binary Vector ◽

Adventitious Shoots ◽

Leaf Explants ◽

Vaccinium Macrocarpon ◽

Marker Genes ◽

Regeneration Medium ◽

Selectable Marker Genes ◽

Gus Gene Expression

Putative transgenic cranberry plants have been achieved via Agrobacterium-mediated transformation. Leaf explants were transformed with a supervirulent Agrobacterium tumefaciens strain EHA 105, harboring the binary vector P35SGUSint and nptII selectable marker genes. Inoculation of precultured explants (≈10 days on regeneration medium) coupled with sonicasion improved transformation efficiency significantly. Adventitious shoots were directly regenerated from explants. Putative transformed shoots were identified by being kanamycin-resistant and GUS-positive. Stable GUS gene expression (turning blue) could be detected within 1 h of incubation at 37 °C. Confirmation of transformation by molecular analysis is in progress. Eight putative transgenic cranberry plants were obtained. All appeared morphologically normal. This appears to be the first success in achieving cranberry transformed plants by Agrobacterium-mediated method. Optimizing the transformation system is ongoing.

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Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Genes ◽

10.3390/genes10050374 ◽

2019 ◽

Vol 10 (5) ◽

pp. 374 ◽

Cited By ~ 9

Author(s):

Dengxiang Du ◽

Ruchang Jin ◽

Jinjie Guo ◽

Fangdong Zhang

Keyword(s):

Transgenic Plants ◽

Selectable Marker ◽

Marker Gene ◽

Transformation Process ◽

Transgenic Maize ◽

Vector System ◽

Marker Genes ◽

Site Specific ◽

Selectable Marker Genes ◽

Marker Free

Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

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Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

Functional Plant Biology ◽

10.1071/pp99078 ◽

2000 ◽

Vol 27 (3) ◽

pp. 201 ◽

Cited By ~ 15

Author(s):

Narayana M. Upadhyaya ◽

Brian Surin ◽

Kerrie Ramm ◽

Judy Gaudron ◽

Petra H. D. Schünmann ◽

...

Keyword(s):

Selectable Marker ◽

Binary Vector ◽

Marker Gene ◽

Gus Activity ◽

Hygromycin Resistance ◽

Rice Cultivars ◽

Selectable Marker Gene ◽

Ubiquitin Promoter ◽

Camv35s Promoter ◽

Derivatives Of

We report the first successfulAgrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph(encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4- fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 car-rying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher ß-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity com-parable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activ-ity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

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Genetic transformation of micropropagated shoots ofPinus radiataD.Don

10.1101/030080 ◽

2015 ◽

Cited By ~ 2

Author(s):

Jan E Grant ◽

Pauline A Cooper ◽

Tracy M Dale

Keyword(s):

Agrobacterium Tumefaciens ◽

Selectable Marker ◽

Marker Gene ◽

Radiata Pine ◽

Commercial Application ◽

Bar Gene ◽

Selectable Marker Gene ◽

Transgenic Lines ◽

Field Deployment ◽

And Control

Abstract Key MessageAgrobacterium tumefacienswas used to transform radiata pine shoots and to efficiently produce stable genetically modified pine plants. Abstract Micropropagated shoot explants fromPinus radiataD. Don were used to produce stable transgenic plants byAgrobacterium tumefaciensmediated transformation. Using this method any genotype that can be micropropagated could produce stable transgenic lines. As over 80% ofP. radiatagenotypes tested can be micropropagated, this effectively means that any line chosen for superior characteristics could be transformed. There are well established protocols for progressing such germplasm to field deployment. Here we used open and control pollinated seed lines and embryogenic clones. The method developed was faster than other methods previously developed using mature cotyledons. PCR positive shoots could be obtain within 6 months ofAgrobacteriumcocultivation compared with 12 months for cotyledon methods. Transformed shoots were obtained using either kanamycin or geneticin as the selectable marker gene. Shoots were recovered from selection, were tested and were not chimeric, indicating that the selection pressure was optimal for this explant type. GFP was used as a vital marker, and the bar gene, (for resistance to the herbicide Buster®/Basta®) was used to produce lines that could potentially be used in commercial application. As expected, a range of expression phenotypes were identified for both these reporter genes and the analyses for expression were relatively easy.

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