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Gateway-compatible tissue-specific vectors for plant transformation.

Michniewicz M, Frick EM, Strader LC - BMC Res Notes (2015)

Bottom Line: To allow for cloning of presumptive promoters with tissue-specific activities, we created two plant expression vectors with multiple cloning sites upstream of a Gateway cassette for expression of either untagged or YFP-tagged genes of interest.For fast and easy tissue-specific expression of desired genes, we further developed an initial set of Gateway-compatible tissue-specific gene expression vectors that allow for the expression of YFP-tagged or untagged proteins driven by the ALCOHOL DEHYDROGENASE1, CHLOROPHYLL A/B BINDING PROTEIN 1, COBRA LIKE1, EXPANSIN7, LATERAL ORGAN BOUNDARIES-DOMAIN 16, SCARECROW, UBIQUITIN10, and WOODEN LEG upstream regulatory regions.These vectors provide an invaluable resource to the plant community, allowing for rapid generation of a variety of tissue-specific expression constructs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Washington University, St. Louis, MO, 63130, USA. martampaciorek@gmail.com.

ABSTRACT

Background: Understanding regulation of developmental events has increasingly required the use of tissue-specific expression of diverse genes affecting plant growth and environmental responses.

Findings: To allow for cloning of presumptive promoters with tissue-specific activities, we created two plant expression vectors with multiple cloning sites upstream of a Gateway cassette for expression of either untagged or YFP-tagged genes of interest. For fast and easy tissue-specific expression of desired genes, we further developed an initial set of Gateway-compatible tissue-specific gene expression vectors that allow for the expression of YFP-tagged or untagged proteins driven by the ALCOHOL DEHYDROGENASE1, CHLOROPHYLL A/B BINDING PROTEIN 1, COBRA LIKE1, EXPANSIN7, LATERAL ORGAN BOUNDARIES-DOMAIN 16, SCARECROW, UBIQUITIN10, and WOODEN LEG upstream regulatory regions.

Conclusions: These vectors provide an invaluable resource to the plant community, allowing for rapid generation of a variety of tissue-specific expression constructs.

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Related in: MedlinePlus

pMCS:YFP-GW and pMCS:GW plant transformation vectors. pMCS:YFP-GW and pMCS:GW were derived from the pEarleyGate104 [12] and pEarleyGate100 [12] vectors, respectively. The pMCS:YFP-GW and pMCS:GW vectors are binary vectors for plant transformation and confer kanamycin (Km, red arrow) and chloramphenicol (in Gateway cassette) resistance in Escherichia coli and kanamycin resistance (Km, red arrow) in Agrobacterium tumefaciens. Plants transformed with these vectors will display resistance to phosphinothricin (Basta; BAR consisting of the basta resistance gene driven by the mannopine synthase promoter and flanked by the mannopine synthase 3′ end; three green arrows). The multiple cloning site (MCS) allows for cloning of desired promoters for expression of downstream genes transferred into the vector using Gateway technology. The Gateway cassette (attR1, chloramphenicol resistance gene, ccdB, attR2; pink arrow) is followed by the terminator sequence from the octopine synthase gene (OCS, blue arrow). In addition, pMCS:YFP-GW has the yellow fluorescence protein (YFP, yellow arrow) gene downstram of the MCS and in-frame with the Gateway cassette. The left border (LB) and right border (RB) of the T-DNA are marked.
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Fig1: pMCS:YFP-GW and pMCS:GW plant transformation vectors. pMCS:YFP-GW and pMCS:GW were derived from the pEarleyGate104 [12] and pEarleyGate100 [12] vectors, respectively. The pMCS:YFP-GW and pMCS:GW vectors are binary vectors for plant transformation and confer kanamycin (Km, red arrow) and chloramphenicol (in Gateway cassette) resistance in Escherichia coli and kanamycin resistance (Km, red arrow) in Agrobacterium tumefaciens. Plants transformed with these vectors will display resistance to phosphinothricin (Basta; BAR consisting of the basta resistance gene driven by the mannopine synthase promoter and flanked by the mannopine synthase 3′ end; three green arrows). The multiple cloning site (MCS) allows for cloning of desired promoters for expression of downstream genes transferred into the vector using Gateway technology. The Gateway cassette (attR1, chloramphenicol resistance gene, ccdB, attR2; pink arrow) is followed by the terminator sequence from the octopine synthase gene (OCS, blue arrow). In addition, pMCS:YFP-GW has the yellow fluorescence protein (YFP, yellow arrow) gene downstram of the MCS and in-frame with the Gateway cassette. The left border (LB) and right border (RB) of the T-DNA are marked.

Mentions: We created the pMCS:GW and pMCS:YFP-GW vectors from pEarleyGate100 and pEarleyGate104 by replacing the 35S promoter regions of these starting vectors with a multiple cloning site (MCS). Because the EcoRI and XhoI sites flanking the 35S promoter are not unique in pEarleyGate100 and pEarleyGate104, we used site-directed mutagenesis to first remove the EcoRI site from the chloramphenicol resistance gene in the Gateway cassette of pEarleyGate100 and pEarleyGate104 and to remove the XhoI and EcoRI sites from the YFP gene of pEarlyGate104, while retaining the correct translation for the chloramphenicol resistance gene and YFP. We then excised the 35S promoter from the mutated versions of pEarleyGate100 and pEarleyGate104 using EcoRI and XhoI and replaced it with a MCS providing EcoRI, NruI, AatII, PmlI, and XhoI sites for cloning promoters of interest. We named these vectors pMCS:GW and pMCS:YFP-GW (Figure 1).Figure 1


Gateway-compatible tissue-specific vectors for plant transformation.

Michniewicz M, Frick EM, Strader LC - BMC Res Notes (2015)

pMCS:YFP-GW and pMCS:GW plant transformation vectors. pMCS:YFP-GW and pMCS:GW were derived from the pEarleyGate104 [12] and pEarleyGate100 [12] vectors, respectively. The pMCS:YFP-GW and pMCS:GW vectors are binary vectors for plant transformation and confer kanamycin (Km, red arrow) and chloramphenicol (in Gateway cassette) resistance in Escherichia coli and kanamycin resistance (Km, red arrow) in Agrobacterium tumefaciens. Plants transformed with these vectors will display resistance to phosphinothricin (Basta; BAR consisting of the basta resistance gene driven by the mannopine synthase promoter and flanked by the mannopine synthase 3′ end; three green arrows). The multiple cloning site (MCS) allows for cloning of desired promoters for expression of downstream genes transferred into the vector using Gateway technology. The Gateway cassette (attR1, chloramphenicol resistance gene, ccdB, attR2; pink arrow) is followed by the terminator sequence from the octopine synthase gene (OCS, blue arrow). In addition, pMCS:YFP-GW has the yellow fluorescence protein (YFP, yellow arrow) gene downstram of the MCS and in-frame with the Gateway cassette. The left border (LB) and right border (RB) of the T-DNA are marked.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig1: pMCS:YFP-GW and pMCS:GW plant transformation vectors. pMCS:YFP-GW and pMCS:GW were derived from the pEarleyGate104 [12] and pEarleyGate100 [12] vectors, respectively. The pMCS:YFP-GW and pMCS:GW vectors are binary vectors for plant transformation and confer kanamycin (Km, red arrow) and chloramphenicol (in Gateway cassette) resistance in Escherichia coli and kanamycin resistance (Km, red arrow) in Agrobacterium tumefaciens. Plants transformed with these vectors will display resistance to phosphinothricin (Basta; BAR consisting of the basta resistance gene driven by the mannopine synthase promoter and flanked by the mannopine synthase 3′ end; three green arrows). The multiple cloning site (MCS) allows for cloning of desired promoters for expression of downstream genes transferred into the vector using Gateway technology. The Gateway cassette (attR1, chloramphenicol resistance gene, ccdB, attR2; pink arrow) is followed by the terminator sequence from the octopine synthase gene (OCS, blue arrow). In addition, pMCS:YFP-GW has the yellow fluorescence protein (YFP, yellow arrow) gene downstram of the MCS and in-frame with the Gateway cassette. The left border (LB) and right border (RB) of the T-DNA are marked.
Mentions: We created the pMCS:GW and pMCS:YFP-GW vectors from pEarleyGate100 and pEarleyGate104 by replacing the 35S promoter regions of these starting vectors with a multiple cloning site (MCS). Because the EcoRI and XhoI sites flanking the 35S promoter are not unique in pEarleyGate100 and pEarleyGate104, we used site-directed mutagenesis to first remove the EcoRI site from the chloramphenicol resistance gene in the Gateway cassette of pEarleyGate100 and pEarleyGate104 and to remove the XhoI and EcoRI sites from the YFP gene of pEarlyGate104, while retaining the correct translation for the chloramphenicol resistance gene and YFP. We then excised the 35S promoter from the mutated versions of pEarleyGate100 and pEarleyGate104 using EcoRI and XhoI and replaced it with a MCS providing EcoRI, NruI, AatII, PmlI, and XhoI sites for cloning promoters of interest. We named these vectors pMCS:GW and pMCS:YFP-GW (Figure 1).Figure 1

Bottom Line: To allow for cloning of presumptive promoters with tissue-specific activities, we created two plant expression vectors with multiple cloning sites upstream of a Gateway cassette for expression of either untagged or YFP-tagged genes of interest.For fast and easy tissue-specific expression of desired genes, we further developed an initial set of Gateway-compatible tissue-specific gene expression vectors that allow for the expression of YFP-tagged or untagged proteins driven by the ALCOHOL DEHYDROGENASE1, CHLOROPHYLL A/B BINDING PROTEIN 1, COBRA LIKE1, EXPANSIN7, LATERAL ORGAN BOUNDARIES-DOMAIN 16, SCARECROW, UBIQUITIN10, and WOODEN LEG upstream regulatory regions.These vectors provide an invaluable resource to the plant community, allowing for rapid generation of a variety of tissue-specific expression constructs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Washington University, St. Louis, MO, 63130, USA. martampaciorek@gmail.com.

ABSTRACT

Background: Understanding regulation of developmental events has increasingly required the use of tissue-specific expression of diverse genes affecting plant growth and environmental responses.

Findings: To allow for cloning of presumptive promoters with tissue-specific activities, we created two plant expression vectors with multiple cloning sites upstream of a Gateway cassette for expression of either untagged or YFP-tagged genes of interest. For fast and easy tissue-specific expression of desired genes, we further developed an initial set of Gateway-compatible tissue-specific gene expression vectors that allow for the expression of YFP-tagged or untagged proteins driven by the ALCOHOL DEHYDROGENASE1, CHLOROPHYLL A/B BINDING PROTEIN 1, COBRA LIKE1, EXPANSIN7, LATERAL ORGAN BOUNDARIES-DOMAIN 16, SCARECROW, UBIQUITIN10, and WOODEN LEG upstream regulatory regions.

Conclusions: These vectors provide an invaluable resource to the plant community, allowing for rapid generation of a variety of tissue-specific expression constructs.

Show MeSH
Related in: MedlinePlus