Soil salinity can be a limiting factor for productivity in agriculture and forestry. In order to fully utilize saline lands productively in plantation forestry for pulp production, the genetic modification of tree species for salt-tolerance may be required. The AhDREB1 gene, a DREB-like transcription factor gene, was transferred into Populus tomentosa by Agrobacterium-mediated transformation. Transgenic plants were regenerated and selected using a two-step process; first on Murashige and Skoog (MS) basal medium containing 4.44 μM 6-benzyladenine (BA), 1.61 μM α-naphthaleneacetic acid (NAA), 30 mg l−1 kanamycin, and 250 mg l−1 ceftomine, and then enhanced selection on medium with 50 mg l−1 kanamycin. The putative transformants were confirmed by polymerase chain reaction (PCR) and Southern hybridization for the AhDREB1 gene. Transgenic plants were rooted on half-strength MS medium containing 5.71 μM indole-3-acetic acid (IAA), 1.61 μM NAA, 2 g l−1 sucrose, and 5 g l−1 agar. The salt tolerance of transgenic plants in pots in the greenhouse showed a survival rate of 100, 100, 84.4, and 44.4% after watering with a solution of 34.2, 68.4, 102.7, and 136.9 mM NaCl, respectively.

https://www.fs.fed.us/nrs/pubs/jrnl/2012/nrs_2012_du_001.pdf

Genetic transformation of Populus tomentosa to improve salt tolerance

Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes.

Research advances on transgenic plant vaccines.

The lignans of Eucommia ulmoides have been extensively studied and shown to have a dual mechanism of regulating blood pressure Studies have shown that DIR1 is a key gene in the biosynthetic pathway of lignans in Eucommia ulmoides Oliv In this study, a 2000 bp upstream promoter sequence was cloned, and part of the sequence (1495 bp) and its 5′-end truncated segment were constructed into the pCAMBIA1391Z expression plasmid upstream of s-glucuronidase (GUS) Agrobacterium-mediated genetic transformation produced stable transgenic tobacco lines The results showed that although both full-length and truncated promoters could initiate GUS expression, their levels were influenced by the degree of deletion at the 5′ end GUS histochemical staining showed that the core promoter region was located in the region containing the transcription initiation site (TIS) within 212 bp In addition, the DIR1 promoter responded to environmental and hormonal stressors, such as jasmonic acid (MeJA), abscisic acid (ABA), d-mannitol (drought mimic), and high concentrations of NaCl In transgenic tobacco seedlings, MeJA, d-mannitol, and ABA could activate the DIR1 promoter, whereas high concentrations of NaCl could inhibit it In E ulmoides Oliv seedlings, MeJA, NaCl, and d-mannitol activated the DIR1 promoter, whereas ABA had an inhibitory effect In summary, our findings provide a theoretical basis for the use of the DIR1 promoter in plant genetic engineering, indicating its potential Our study also presents novel insights for lignan biosynthesis and sheds light on the mechanisms of E ulmoides Oliv in response to stress We successfully cloned the DIR1 gene promoter and studied its function in regulating the expression of the DIR1 gene

/pdf/cloning-and-characterization-of-the-dir1-promoter-from-3k5taw9fz7.pdf

Cloning and characterization of the DIR1 promoter from Eucommia ulmoides Oliv and its response to hormonal and abiotic stress

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

Review on plant gene promoters

Simultaneous introduction of multiple genes into plants is a critical step in plant genetic engineering to manipulate multiple functional genes in metabolic engineering and trait stacking. It is important to construct a bidirectional promoter for transforming two or more genes into plants simultaneously. The widely used unidirectional CaMV35S promoter has been modified to a bidirectional promoter in this work by fusing a CaMV35S minimal promoter (Pmini) at its end in opposite orientation to the original promoter. To test its bi-directional transcriptional activities, two widely used histochemically visible reporter genes, gusA (β-glucuronidase) from Escherichia coli and gfp (Green Fluorescent Protein) from Aequorea victoria, were fused to the terminus of the bidirectional promoter in different orientations ending with NOS terminator sequences. The transient expression of the gusA and gfp genes were detected by histochemical staining for GUS and by fluorescence microscopy for GFP. The direction of transient expression of GUS and GFP in Agrobacterium mediated 3–4 days transformed leaf discs of Populus tomentosa, indicating that the promoter did have bidirectional transcriptional activities simultaneously in cells and tissues. It was discussed that this bidirectional promoter could possibly be applied in woody plant engineering.

Construction of a bidirectional promoter and its transient expression in Populus tomentosa

The invention discloses a bidirectional promoter bi-visual fluorescent protein report gene plant expression vector. The promoter bi-visual fluorescent protein report gene plant expression vector is a recombinant expression vector of which the multiple cloning sites of an initial vector are inserted with the following DNA molecules: an EcoRI enzyme recognition sequence, an NoS terminator, an SacI enzyme recognition sequence, a kpnI enzyme recognition sequence, a red fluorescent protein gene, an SmalI enzyme recognition sequence, a BamHI enzyme recognition sequence, an XbalI enzyme recognition sequence, a bidirectional promoter, an XholI enzyme recognition sequence, an SpeI enzyme recognition sequence, a green fluorescent protein, an HindIII enzyme recognition sequence, an HpaI enzyme recognition sequence, an SalI enzyme recognition sequence, an NoS terminator and a Clal contained in turn from 5' end; and the nucleotide sequence of the bidirectional promoter is from the 5917th site to the 6834th site from the 5' end in a sequence 1 of a sequence list. The bidirectional promoter bi-visual fluorescent protein report gene plant expression vector can be applied to fusing functional genes by simple operation of direct enzyme cutting and connection.

Bidirectional promoter bi-visual fluorescent protein report gene plant expression vector

The invention aims at providing a two-way starting plant expression vector system of double recombination sites. The two-way starting plant expression vector system is composed of a two-way expressing plant expression vector pRGFL of double recombination sites and two cloning vectors p-FRT and p-loxp with specific recombination sites respectively. The two-way expressing plant expression vector pRGFL of the double recombination sites provided by the invention is a transformed plant expression vector containing specific DNA molecules. The two cloning vectors with the specific recombination sites respectively provided by the invention are respectively inserted into the recombination vector containing the specific DNA molecules at multiple cloning sites of a starting vector. The cloning vector p-FRT and the cloning vector p-loxp provided by the invention form a vector with a T tail end or a flat tail end after being digested by XcmI enzyme or EcorV enzyme, and can be directly connected with a PCR product of a target gene to achieve the cloning target. The target genes on the cloning vector p-FRT and the cloning vector p-loxp can respectively replace fluorescence protein reporter genes on the two-way expressing plant expression vector of the double recombination sites through specific site combination, and thus the target genes are transferred to the plant expression vector; only enzyme medium needs to be recombined in the process; and other operations are not required, so that the two-way starting plant expression vector system is simple and feasible.

Two-way starting plant expression vector system of double recombination sites

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Review on plant gene promoters

Construction of a bidirectional promoter and its transient expression in Populus tomentosa

Bidirectional promoter bi-visual fluorescent protein report gene plant expression vector

Two-way starting plant expression vector system of double recombination sites