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First report about the identification and preliminary analysis of a partial sequence of dihydropyrimidine dehydrogenase (NADP+) in Thermopsis turcica during floral development using degenerate primers

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Abstract

Thermopsis turcica Kit Tan, Vural & Küçüködük is an herbaceous perennial, endemic and listed as endangered species by IUCN in Turkey. This plant is noted for its unusual floral structure characterized by a 2–4 carpellary ovary. In this study, a DPD (NADP+)-like gene was partially sequenced for the first time in T. turcica. Since there is no previous molecular genetic information available for T. turcica, RT-PCR was performed using degenerate primers targeted to conserved sequences of WUS protein homologues from related legume species. Amplified cDNAs of the expected size were sequenced and analyzed using bioinformatics tools. The analysis strongly suggested that a 283 bp PCR product was part of a dihydropyrimidine dehydrogenase (NADP+)-like coding sequence with a Flavin mononucleotide binding domain. The putative gene was named Tt-DPD and the partial CDS submitted to the NCBI database (accession number KT182937). This gene has not been identified previously in T. turcica. The DPD (NADP+) enzyme is the rate-limiting step in pyrimidine degradation, which is essential for the biosynthesis of beta-alanine and pantothenates in plants; it has also been shown to be required for normal seed development in Arabidopsis. Expression of Tt-DPD was monitored by both endpoint and real-time RT-PCR. High expression of the identified gene was observed in the mid-developmental stage of the pistil of T. turcica. The findings presented here provide a starting point for understanding the roles of this gene in pyrimidine catabolism in T. turcica.

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Abbreviations

Aa:

Amino acid(s)

AGE:

Agarose gel electrophoresis

BLAST:

Basic local alignment search tool

bp:

Base pair

C. arietinum :

Cicer arietinum

cDNA:

Complementary deoxyribonucleic acid

CDS:

Coding sequence

DPD:

Dihydropyrimidine dehydrogenase

5-FU:

5-Fluorouracil

L. japonicus :

Lotus japonicus

M. truncatula :

Medicago truncatula

NADP:

Nicotinamide-adenine dinucleotide phosphate

NCBI:

National Center for Biotechnology Information

NGBG:

Nezahat Gökyiğit Botanical Garden

ORF:

Open reading frame

PCR:

Polymerase chain reaction

P. vulgaris :

Phaseolus vulgaris

RT-PCR:

Reverse transcription polymerase chain reaction

TBE:

Tris–boric acid–EDTA buffer

T. turcica :

Thermopsis turcica

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Acknowledgements

The authors express their appreciation to Nezahat Gökyiğit Botanical Garden for providing research materials for the study. The authors are grateful to Ali Nihat Gökyiğit and Prof.Dr. Adil Güner (Nezahat Gökyiğit Botanical Garden) for their support. The assistance of Burçin Çıngay (Garden Department of Nezahat Gökyiğit Botanical Garden) is greatly appreciated. This work was supported by the Ali Nihat Gökyiğit Foundation of Turkey.

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Authors and Affiliations

  1. Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli-Tuzla, 34956, Istanbul, Turkey

    D. Tekdal & S. Cetiner

  2. Nanotechnology Research and Application Centre, Sabanci University, Orhanli-Tuzla, 34956, Istanbul, Turkey

    S. J. Lucas

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  1. D. Tekdal
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Correspondence to D. Tekdal.

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We declare that we have no conflict of interest. We certify that all persons who have made substantial contributions to this manuscript. We certify that the submitted original manuscript is not under review by any other journal. There is no financial interest to report.

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Communicated by P. K. Nagar.

Electronic supplementary material

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11738_2017_2458_MOESM1_ESM.tiff

Fig. S1. Pairwise sequence alignment score between T. turcica and representative plant legumes; French bean (P. vulgaris), chickpea (C. arietinum), and barrel medic (M. truncatula) regarding DPD (NADP +) gene amplified by Tt-DPD primer pairs. Species and their GenBank accession numbers are described in Table 1 in the paper (TIFF 1428 kb)

11738_2017_2458_MOESM2_ESM.tiff

Fig. S2. Comparison of amino acid sequences of DPD (NADP +) in T. turcica and other representative plant legumes; French bean (P. vulgaris), chickpea (C. arietinum), and barrel medic (M. truncatula) using Muscle 3.8.31 program (http://www.phylogeny.fr/one_task.cgi?task_type=muscle). Identical and conserved amino acids are shaded. Species and their GenBank accession numbers are described in Table 1 in the paper (TIFF 991 kb)

11738_2017_2458_MOESM3_ESM.tiff

Fig. S3. DPD gene homologues used for multiple alignment and phylogenetic tree. ‘*,’ Identical amino acid residues (TIFF 4337 kb)

11738_2017_2458_MOESM4_ESM.tiff

Fig. S4. All fragments which cover the same region of the homologue gene were used for sequence analyzing (TIFF 9151 kb)

Fig. S5. Conceptual translation of the ORFs of the sequences described in Figure S4 (TIFF 1692 kb)

11738_2017_2458_MOESM6_ESM.tiff

Fig. S6. Comparison of amino acid sequences of DPD in Thermopsis turcica with the representative gene in Cicer arietinum using the technique mentioned in the paper (TIFF 1314 kb)

Fig. S7. Putative conserved domains in DPD protein in T. turcica (TIFF 394 kb)

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Tekdal, D., Lucas, S.J. & Cetiner, S. First report about the identification and preliminary analysis of a partial sequence of dihydropyrimidine dehydrogenase (NADP+) in Thermopsis turcica during floral development using degenerate primers. Acta Physiol Plant 39, 159 (2017). https://doi.org/10.1007/s11738-017-2458-x

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