[Show abstract][Hide abstract] ABSTRACT: We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71 bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.
No preview · Article · Dec 2011 · DNA and cell biology
[Show abstract][Hide abstract] ABSTRACT: We isolated a DREB orthologue, MtDREB1C, from Medicago truncatula. Its deduced protein contains an AP2 domain of 57 amino acids. Yeast one-hybrid assay revealed that MtDREB1C specifically
bound to the dehydration-responsive element (DRE) and activated the expression of HIS3 and LacZ reporter genes. In a transcriptional activation assay, coexpression of the MtDREB1C cDNA resulted in much higher (21.2 times) transactivation of the LacZ reporter gene than experiments performed without MtDREB1C. Transformation of Medicago revealed that overexpression of MtDREB1C suppressed shoot growth, and enhanced the freezing tolerance of M. truncatula. The MtDREB1C gene was transformed into China Rose (Rosa chinensis Jacq.) driven by Arabidopsis
rd29A promoter. Southern-blot analysis showed that the target gene was integrated into the genome of a surviving transgenic rose
plant. Northern-blot analysis illustrated that robust expression of MtDREB1C was only activated under stress conditions, and the expressed MtDREB1C mRNA reached maximum accumulation 10h following freezing treatment. The performance of the transgenic line under freezing
stress was superior to untransformed controls. This transgenic plant continued to grow, flowered under unstressed conditions,
and was phenotypically normal. These facts indicate that the MtDREB1C gene, isolated from Medicago truncatula and driven by the Arabidopsis rd29A promoter, enhanced freezing tolerance in transgenic China Rose significantly without any obvious morphological or developmental
KeywordsChina rose-DRE-binding-Freezing tolerance-
[Show abstract][Hide abstract] ABSTRACT: Here we describe a rapid and efficient PCR-mediated ligation protocol for constructing a plant RNA interference vector to express long hairpin RNA (hpRNA). In the protocol, four oligonucleotide primers were used and three rounds of PCRs performed. The product of the first PCR was used as a megaprimer for the second PCR to generate a chimeric molecule with a gene-specific sequence and a spacer spliced together. The chimeric product could be used as another megaprimer for the third PCR to ligate another gene-specific sequence to the other end of the spacer, but in the reverse orientation. Thus, within a few days, two gene-specific sequences could be ligated to a spacer in the antisense and sense orientations using the PCR-mediated ligation method, without reliance on restriction cleavage and DNA ligation. The ligated product could be inserted into the plant expression vector for plant transformation. The transcribed RNA formed hpRNA constructs containing sense/antisense arms for specific gene targeting. Overexpression of hpRNA constructed by a Medicago truncatula xyloglucan endotransglycosylase gene retarded the growth of transgenic M. truncatula roots.
No preview · Article · Sep 2009 · DNA and cell biology
[Show abstract][Hide abstract] ABSTRACT: We isolated a DREB homologue gene, MtDREB2A, from Medicago truncatula. Its deduced protein contains an AP2 domain of 59 amino acids. The expression of MtDREB2A was significantly induced in roots by salt and drought treatments. Using megaprimer PCR, we deleted a Ser/Thr-rich coding
region between residues 142 and 190, and transformed MtDREB2A to a constitutive form, namely, MtDREB2a. Yeast one-hybrid assay
revealed that both MtDREB2A and MtDREB2a specifically bound to the dehydration-responsive element (DRE) and activated the
expression of the reporter genes of HIS3 and LacZ. Analysis of transcription activities of the proteins in yeast indicated that MtDREB2a could activate the expression of reporter
gene, whereas MtDREB2A could not. Overexpression of MtDREB2a in transgenic M. truncatula resulted in significant dwarfed seedling. These results suggested that MtDREB2A functioned specially in response to salt and drought stresses in M. truncatula and that deletion of the Ser/Thr-rich region between residues 142 and 190 activated the transcriptional activation ability
No preview · Article · Feb 2009 · In Vitro Cellular & Developmental Biology - Plant
[Show abstract][Hide abstract] ABSTRACT: Megaprimer-based methodology has been widely applied in site-directed mutagenesis, but rarely used in gene splicing. In this article, we describe a modification of the megaprimer PCR method, which can efficiently create and amplify a specific ligated chimeric gene segment in a PCR reaction and under a common PCR program that is widely used by researchers. More importantly, this modified method for splicing two or more gene fragments together revealed the mechanism of the megaprimer PCR method, by elucidating the key factor in the megaprimer-based protocol. In this method, the denatured megaprimer divided into two strands. One strand was used as template DNA to regenerate megaprimer and the other strand was used as an oligonucleotide primer to create a ligated chimeric gene product. In this article, we detail the modified megaprimer protocol for creating and amplifying these chimeric gene products, including a specific protocol for large chimeric gene products. We also provide additional tips to increase specificity and efficiency of the protocols. In conclusion, the improved megaprimer PCR protocol is a simple, broadly applicable protocol for splicing two different gene fragments together without relying on restriction sites.
No preview · Article · Jul 2008 · Molecular Biotechnology