УДК 581.1
PCR Primers Targeting сis-Acting Promoter Elements in Plants1
© 2012 M. Zhao, Z. Liu, S. Chen, F. Chen, J. Jiang, A. Song
College of Horticulture, Nanjing Agricultural University, Nanjing, China
Received May 25, 2011
The cis-acting elements present in gene promoters are important for promoter function. Thermal Asymmetric Interlaced PCR (TAIL-PCR) provides a simple means for isolating promoter sequences, but the necessary primers can be troublesome to design. Here, we describe an approach, which targets cis-acting elements for TAIL-PCR. The method combines the advantages of TAIL-PCR and SiteFinding-PCR. The new method proved successful for isolating a number of plant promoter sequences.

Keywords: plant  primer design  cis-acting elements  TAIL-PCR

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1 This text was submitted by the authors in English.
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Abbreviations: AD  arbitrary degenerate; CE  cis-acting element; TAIL-PCR  Thermal Asymmetric Interlaced PCR.
Corresponding address: Fadi Chen. College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China. Fax: �25-8439-5266; e-mail: chenfd@njau.edu.cn
INTRODUCTION
The promoter is an indispensable part of a functional gene; its sequence includes a variety of cis-acting elements, which are responsible for the promoter binding with transcription factors [1], and this interaction then determines the level of both temporal and spatial expression of the gene. Promoters have been classified into those, which drive constitutive expression, those, which allow the gene to be induced, and those, which are either tissue- or developmental stage-specific . Constitutive promoters are associated with genes expressed throughout the life cycle and throughout the plant, while inducible ones respond to the presence of a particular physical or chemical cue. The latter type has been exploited in certain genetic engineering applications [3, 4]. The constitutive 35S cauliflower mosaic virus (35S CaMV) promoter is effective in many plant species and has been much used for the investigation of transgenic function [5]. Despite promoter diversity, they share a number of core elements, in particular the TATA CAAT boxes [6]. Once a promoter sequence has been determined, its functionality can be assessed in a number of ways [7, 8]. Several PCR-based methods have been developed to isolate promoter sequences, and one of the most effective of these is TAIL-PCR [9]. Its advantage lies in its simplicity, specificity, and efficiency. A more recent method, termed SiteFinding-PCR, was suggested by Tan et al. [10]; however, less effective TAIL-PCR provides a simpler means for designing the necessary primers. Here, we describe a modification of TAIL-PCR, which combines the advantageous features of TAIL-PCR and SiteFinding-PCR.

MATERIALS AND METHODS
Genomic DNA was extracted from the leaves of chrysanthemum, using a conventional CTAB method [11]. The template DNA had an A260/A280 ratio of ~1.8 and was diluted to 3050 ng/ml. Three genes were chosen from each of Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa, Physcomitrella patens, Populus trichocarpa, Sorghum bicolor, Vitis vinifera, and Zea mays (table 1). A 3-kb stretch upstream of the transcription start site of each gene was retrieved from the ENSEMBL sequence database (http://plants.ensembl.org/info/about/species.html) to ensure that the promoter was included. A second 3-kb stretch lying 2730 kb upstream of the transcription start site of each gene was also retrieved to act as a control. PLACE software (www.dna.affrc.go.jp/PLACE/signalscan.html) was employed to identify the cis-acting elements present. If the number of a motif existed in single sequence was beyond 20 or below 3, the motif was not involved in primer design in the experimental group. Then the selected motifs were compared in the experimental group to indentify what were existed commonly, which means the motif presented in > 10 of 24 genes. The conclusive chosen motifs were targeted for PCR primer design (fig. 1). An adapter sequence plus 23 extra bases was added to the 5' end of the primer targeting a motif sequence (fig. 1). Primer sequences were designed using primer v. 5.0 software (“Premier Biosoft”, Palo Alto, United States) (table 2). A nested three step PCR strategy was used to amplify the target promoter sequences. AD primers were chosen as a positive control.
Genomic DNA represented the template for the primary TAIL-PCR, and the resulting amplicon was diluted 50100 times to form the template for the secondary TAIL-PCR. The PCR programs would run by the thermal condition of TAIL-PCR (table 3), while in the tertiary PCR, for the experimental group, a general PCR program (30 cycles of 94°C/30s, 50°C/60s, 72°C/120s) was applied, because CE primers had a consensus sequence adapted to AC primer [10]. The composition of each PCR is given in table 4. The tertiary PCR products were separated electrophoretically on 1% agarose gels.

RESULTS
The comparison of the amplicons showed the disparities between the CE and AD groups (fig. 2). All primers listed in table 2 were usable for TAIL-PCR. Some of the primers (groups 1 and 2) were as effective as the AD primers, while the primes targeting ASF1MOTIFCAMV (TGACG) and BOXIINTPATPB (ATAGAA) failed in the most cases. The length of most amplicons was > 500 bp and frequently more than one distinct sequence was amplified.

DISCUSSION
Certain primers successfully amplified the template. Those targeting PYRIMIDINEBOXOSRAMY1A (CCTTTT), REALPHALGLHCB21 (AACCAA), and MYBCOREATCYCB1 (AACGG) were more effective than those targeting ASF1MOTIFCAMV (TGACG) or BOXIINTPATPB (ATAGAA). This difference may reflect the absence of particular motifs in the chosen promoters, which was the reason for targeting a range of cis-acting elements. Of the seven primer pairs designed, five proved usable for TAIL-PCR. Given the length of the cloned sequences, the number of motifs present in the promoter was likely not large. Where a particular motif is sited near the transcription start site, the resulting primary amplicon would be too short for the subsequent nesting steps, so targeting POLASIG3 (AATAAT) or TATABOX5 (TTATTT) would not have been sensible for TAIL-PCR. Fortunately, they could amplify the unknown sequences and the length of the sequences was longer than the sequences cloned by AD primers. This may be the result of the TAIL-PCR, which can reduce the unexpected sequences efficiently. However, the sequence context of a motif may also influence the experimental outcome. When PLACE was applied to analyse the promoter, a number of motif types and a variable number of each motif were identified. In some cases, the same motif occurred twice within the same promoter, once in the forward, and once in the reverse direction. Both copies would have been recognized by the relevant primer, and thus unexpected fragments would have been amplified. The TAIL-PCR procedure tends to suppress the amplification of most of these unexpected sequences. While electrophoresis swear occurred in some cases when the AD primers were used, but the use of an AC adapter primer helped reduce the frequency of this phenomenon. In conclusion, primers based on the common primer motif REALPHALGLHCB21 (AACCAA) are suitable as TAIL-PCR AD primers and produce amplicons mostly above 500 bp in length.
This work was supported by the National Natural Science Foundation of China (grant nos. 30872064, 31071820, 31071825) the Fundamental Research Funds for the Central Universities (KYJ 200907), the Program for Hi-Tech Research, Jiangsu, China (grant no. BE2010303), and by Qing Lan Project of Jiangsu Province (grant no. 2008).
REFERENCES
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FIGURE CAPTIONS

Fig. 1. Target motifs for primer design.
The length of the bars indicates the number of promoter motifs present. Sequences shaded in grey are from the upstream region of known genes, while those marked in white derive from sequence 30 kb upstream of the gene transcription start. The motifs can be divided into three groups. In group 1, exemplified by POLASIG, motif frequency was high, and the upstream region group number was bigger than the 30 kb upstream region group. In group 2 (MYBCOREATCYCB), motif frequency was low, but still higher than the 30 kb upstream region group. In group 3, just as ASF1MOTIFCAMV, motif frequency was low and comparable with that found in the 30 kb upstream region group.

Fig. 2. Agarose gel analysis of products of the third step of the nested PCR.
The CE primers run the common PCR, while the AD primers were used for TAIL-PCR. The length of the amplicons was > 500 bp.