Limits...
RMDAP: a versatile, ready-to-use toolbox for multigene genetic transformation.

Ma L, Dong J, Jin Y, Chen M, Shen X, Wang T - PLoS ONE (2011)

Bottom Line: The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system.A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome.This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call "one-stop breeding."

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.

ABSTRACT

Background: The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed.

Methodology/principal findings: Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome.

Conclusions/significance: This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call "one-stop breeding."

Show MeSH

Related in: MedlinePlus

Physical maps of original donor vectors and recipient vectors carrying different marker genes.(A) Schematic illustration of the two original entry vectors pOSB100 and pOSB200, from which the series is derived. The fragment introduced into the pMD18-T simple vector contained a loxP site (blue arrow), a multicloning site (red box), and two rare restriction enzyme sets E1/E2 (underlined). These two sets are in opposite orientation. Ori, pUC replicon; Ampr, ampicillin resistance gene. (B) Construction of binary transformation vectors equipped with no plant selection gene, for marker-free type assembly, or the commonly used plant selection genes of hptII and bar, based on the pCAMBIA family. LB and RB, left border and right border of the T-DNA; HPTII, hygromycin resistance gene cassette; BAR, phosphinothricin resistance gene cassette.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3094388&req=5

pone-0019883-g001: Physical maps of original donor vectors and recipient vectors carrying different marker genes.(A) Schematic illustration of the two original entry vectors pOSB100 and pOSB200, from which the series is derived. The fragment introduced into the pMD18-T simple vector contained a loxP site (blue arrow), a multicloning site (red box), and two rare restriction enzyme sets E1/E2 (underlined). These two sets are in opposite orientation. Ori, pUC replicon; Ampr, ampicillin resistance gene. (B) Construction of binary transformation vectors equipped with no plant selection gene, for marker-free type assembly, or the commonly used plant selection genes of hptII and bar, based on the pCAMBIA family. LB and RB, left border and right border of the T-DNA; HPTII, hygromycin resistance gene cassette; BAR, phosphinothricin resistance gene cassette.

Mentions: First, two original donor vectors, pOSB100 and pOSB200, were constructed (Figure 1A) containing the sequences “loxP+E1+MCS+E2” and “loxP+E2+MCS+E1” respectively (E1 = rare restriction enzyme set 1, MCS = multiple cloning site, E2 = rare restriction enzyme set 2). We chose the pMD18-T simple vector (Takara) as a skeleton with ampicillin resistance, and MCS from pBluescript SKII. Rare restriction enzyme set 1 (E1) contains AscI, SbfI, and homing endonuclease I-PpoI sites, while rare restriction enzyme set 2 (E2) contains PacI, PmeI, and homing endonuclease I-SceI sites. The homing endonucleases are double-stranded DNases with large, asymmetric recognition sites (12–40 bp) (New England BioLabs), so recognition sites are extremely rare. As PacI, PmeI, AscI, and SbfI are the restriction enzymes able to identify eight nucleotide sequences, the occurrence of these is also relatively low, and so they could been carried out identification of the vector by enzyme digestion, and if necessary, also excise the intermediate vector's backbone. All satellite vectors later described are derived from these two original vectors. The RMDAP inherits all of the advantages of the former system [11], [16], but also has greatly expanded functional and utilization aspects. These methods are completely compatible.


RMDAP: a versatile, ready-to-use toolbox for multigene genetic transformation.

Ma L, Dong J, Jin Y, Chen M, Shen X, Wang T - PLoS ONE (2011)

Physical maps of original donor vectors and recipient vectors carrying different marker genes.(A) Schematic illustration of the two original entry vectors pOSB100 and pOSB200, from which the series is derived. The fragment introduced into the pMD18-T simple vector contained a loxP site (blue arrow), a multicloning site (red box), and two rare restriction enzyme sets E1/E2 (underlined). These two sets are in opposite orientation. Ori, pUC replicon; Ampr, ampicillin resistance gene. (B) Construction of binary transformation vectors equipped with no plant selection gene, for marker-free type assembly, or the commonly used plant selection genes of hptII and bar, based on the pCAMBIA family. LB and RB, left border and right border of the T-DNA; HPTII, hygromycin resistance gene cassette; BAR, phosphinothricin resistance gene cassette.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094388&req=5

pone-0019883-g001: Physical maps of original donor vectors and recipient vectors carrying different marker genes.(A) Schematic illustration of the two original entry vectors pOSB100 and pOSB200, from which the series is derived. The fragment introduced into the pMD18-T simple vector contained a loxP site (blue arrow), a multicloning site (red box), and two rare restriction enzyme sets E1/E2 (underlined). These two sets are in opposite orientation. Ori, pUC replicon; Ampr, ampicillin resistance gene. (B) Construction of binary transformation vectors equipped with no plant selection gene, for marker-free type assembly, or the commonly used plant selection genes of hptII and bar, based on the pCAMBIA family. LB and RB, left border and right border of the T-DNA; HPTII, hygromycin resistance gene cassette; BAR, phosphinothricin resistance gene cassette.
Mentions: First, two original donor vectors, pOSB100 and pOSB200, were constructed (Figure 1A) containing the sequences “loxP+E1+MCS+E2” and “loxP+E2+MCS+E1” respectively (E1 = rare restriction enzyme set 1, MCS = multiple cloning site, E2 = rare restriction enzyme set 2). We chose the pMD18-T simple vector (Takara) as a skeleton with ampicillin resistance, and MCS from pBluescript SKII. Rare restriction enzyme set 1 (E1) contains AscI, SbfI, and homing endonuclease I-PpoI sites, while rare restriction enzyme set 2 (E2) contains PacI, PmeI, and homing endonuclease I-SceI sites. The homing endonucleases are double-stranded DNases with large, asymmetric recognition sites (12–40 bp) (New England BioLabs), so recognition sites are extremely rare. As PacI, PmeI, AscI, and SbfI are the restriction enzymes able to identify eight nucleotide sequences, the occurrence of these is also relatively low, and so they could been carried out identification of the vector by enzyme digestion, and if necessary, also excise the intermediate vector's backbone. All satellite vectors later described are derived from these two original vectors. The RMDAP inherits all of the advantages of the former system [11], [16], but also has greatly expanded functional and utilization aspects. These methods are completely compatible.

Bottom Line: The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system.A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome.This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call "one-stop breeding."

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.

ABSTRACT

Background: The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed.

Methodology/principal findings: Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome.

Conclusions/significance: This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call "one-stop breeding."

Show MeSH
Related in: MedlinePlus