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Research Article

Characterization of Thaumatin-like Gene Family and Identification of Pectobacterium carotovorum subsp. carotovorum Inducible Genes in Brassica oleracea

Nasar Uddin Ahmed1, Jong-In Park1, Hee-Jeong Jung1, Mi-Young Chung2, Yong-Gu Cho3, Ill-Sup Nou1,*
Plant Breeding and Biotechnology 2013;1(2):111-121.
Published online: June 30, 2013

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*Corresponding author: Ill-Sup Nou, nis@sunchon.ac.kr, Tel: +82-61-750-3249, Fax: +82-61-750-3208
• Received: May 25, 2013   • Revised: June 17, 2013   • Accepted: June 18, 2013

Copyright © 2013 The Korean Society of Breeding Science

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Characterization of Thaumatin-like Gene Family and Identification of Pectobacterium carotovorum subsp. carotovorum Inducible Genes in Brassica oleracea
Plant Breed. Biotech.. 2013;1(2):111-121.   Published online June 30, 2013
DOI: https://doi.org/10.9787/PBB.2013.1.2.111
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Characterization of Thaumatin-like Gene Family and Identification of Pectobacterium carotovorum subsp. carotovorum Inducible Genes in Brassica oleracea
Plant Breed. Biotech.. 2013;1(2):111-121.   Published online June 30, 2013
DOI: https://doi.org/10.9787/PBB.2013.1.2.111
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Characterization of Thaumatin-like Gene Family and Identification of Pectobacterium carotovorum subsp. carotovorum Inducible Genes in Brassica oleracea
Image Image Image Image
Fig. 1 Phylogenetic tree showing the relatedness of the deduced full-length amino acid sequences of 12 Brassica oleracea thaumatin-like proteins (BoTLPs) and 41 PR thaumatin-like proteins of other plant species. The phylogenetic tree was generated using MEGA5.05 program. The species names and the GenBank protein accessions numbers are indicated in brackets. Brassica oleracea (AAO12209), Brassica rapa (AAB95118), Brassica rapa subsp. chinensis (ACU65941 and BAJ16221), Oryza sativa Japonica Group (BAD30547 and BAC82958), Arabidopsis thaliana (AEE29691, AEE29690, NP001031064, AEE86963, AEE86962, AEE29829, NP173365, AED93336 and AEE29926), Solanum lycopersicum (CAB86199), Nicotiana tabacum (AAW66482, CAA33292 and BAA74546), Zea mays (NP001150507 and NP001151120) and BrTLP1 to 20 were taken from our previous study (Ahmed et al. 2013). The scale represents the frequency of amino acid substitution between sequences as determined by poisson evolutionary distance method.
Fig. 2 Alignment of deduced amino acid sequences of (A) Group I thaumatin-like proteins; (B) Group II thaumatin-like proteins and sweet-tasting thaumatin protein of Thaumatococcus daniellii (Bennett) Benth. (P02884) in each group using PIR. Asterisk marks show all 16 conserved cysteine residues, square bullets indicate REDDD motif in the highly conserved region (rectangle) of the domain and circular bullets indicate FF hydrophobic motif described by Petre et al. (2011) in identical positions among the aligned proteins. Numbers on the right margin indicate the positions of amino acid residues. The identical amino acids are marked in dark background, and more than 50% similarities in light.
Fig. 3 RT-PCR expression analysis of 12 BoTLP genes over different organs at Brassica oleracea plants. Lanes 1–4, PCR products of roots (R), stems (S), leaves (L) and flower buds (Fb)
Fig. 4 Real-time quantitative PCR expression analysis of (A) BoTLP5, (B) BoTLP8 and (C) BoTLP12 genes in Brassica oleracea after infection with Pectobacterium carotovorum subsp. carotovorum (Pcc). The error bars represent the standard error of the means of three independent replicates.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Characterization of Thaumatin-like Gene Family and Identification of Pectobacterium carotovorum subsp. carotovorum Inducible Genes in Brassica oleracea

Primer information of 12 thaumatin-like genes of B. oleracea for RT and Real time PCR.

Gene name Acc. No. of ESTs Primer sequence
Forward Reverse
BoTLP1 BOLC06439 CAGAAATCAACAAGCTACCC GATCCGTCTTGTAACATCGT
BoTLP2 BOLC11620 TTTTCACCATCCAGAATAGC GTCGAAGTTACAGCCTGTTC
BoTLP3 BOLC12781 TCTACCGTTTTCAAGAGAGC CCGTACGGATTCTGAGTTAG
BoTLP4 BOLC13543 CTACTCTCGCAGAGATCACC GAGTTAAAGGCAGAACATGC
BoTLP5 BOLC14356 CCCTGAAAGAAGAATGAGAA ACAAGACGAGCACCTACACT
BoTLP6 BOLC17729 ACGACTTACTCGATGACGTT ATCTTCCGTACATAGCAAGC
BoTLP7 BOLC20473 CCGCAAAAGCTCTTTATTAC TATAGCTACGCATACGACGA
BoTLP8 BOLC20492 ATCCAGATGAGAATCACGAC ACAAGTCTCCGGTTTATTGA
BoTLP9 BOLC23111 GTAGAGACGGGAACAAAACA AAAGTTGCAGTGAAAAGCTC
BoTLP10 BOLC23113 ACGGGAGGATTCTTAATTGT CAACGCAGCATTAGATAACA
BoTLP11 BOLC23820 CGTCCGGATTAATACTTGAG AGTTGTACACCGTTGTCTCC
BoTLP12 BOLS14849 AGCAAAATCCATATCACAGG GACTGTCCCACATCAAGTTT
Br-Actin FJ969844 CAACCAATCGTCTGTGACAA ATGTCTTGGCCTACCAACAA

Overall analysis of thaumatin-like genes collected from the Brassica oleracea database.z)

Name of genes Accession Number ORF (bp) Chromosome number Protein
Length (aa) THN domain Start and end Mol.Wt. (KD) pI
BoTLP1 Bol016062 1041 C03 346 1 to 184 35.58 4.25
BoTLP2 Bol044835 738 C08 245 1 to 185 25.32 5.05
BoTLP3 Bol022411 1098 C07 365 1 to 189 38.56 5.31
BoTLP4 Bol026250 738 C06 245 1 to 181 26.15 9.07
BoTLP5 Bol000789 825 Scaffold 274 1 to 185 29.22 8.91
BoTLP6 Bol026131 732 C06 243 1 to 180 25.63 4.62
BoTLP7 Bol026844 741 C05 246 1 to 184 25.46 4.83
BoTLP8 Bol044836 402 C08 133 1 to 72 14.66 8.29
BoTLP9 Bol027694 978 C06 325 1 to 185, trnsm 1 34.32 4.86
BoTLP10 Bol039281 963 C02 320 1 to 187 33.73 4.67
BoTLP11 Bol026846 747 C05 248 1 to 186 25.88 4.63
BoTLP12 Bol016440 1287 C02 428 1 to 190 45.33 4.95

z)Brassica oleracea database (http://119.97.203.210/bolbase/blast/blast.html).

Homology analysis of 12 thaumatin-like genes of Brassica oleracea z)

Protein Top matched clones Top matched protein Identity Top homologous species References
BoTLP1 NP568046 PR TLPy) 86% Arabidopsis thaliana Mayer et al. (1999)
XP003540838 TLP 65% Glycine max Unpublished
BoTLP2 NP173365 PR TLP 72% A. thaliana Theologis et al. (2000)
AEV57470 TLP1 70% Prunus persica Sherif et al. (2012)
BoTLP3 AED93335 PR TLP 71% A. thaliana Tabata et al. (2000)
XP004309088 TLP1 71% Fragaria vesca subsp. vesca Unpublished
BoTLP4 ABV89611 PR TLP 99% B. rapa Ahmed et al. (2013)
NP177503 PR TLP 96% A. thaliana Theologis et al. (2000)
BoTLP5 NP001078513 PR TLP 93% A. thaliana Mayer et al. (1999)
XP003540840 TLP1 77% G. max Unpublished
BoTLP6 NP177640 TLP3 77% A. thaliana Theologis et al. (2000)
AEV57470 TLP1 73% P. persica Sherif et al. (2012)
BoTLP7 NP173365 PR TLP 71% A. thaliana Theologis et al. (2000)
BAD90814 TLP 66% Cryptomeria japonica Futamura et al. (2006)
BoTLP8 NP180054 PR TLP 74% A. thaliana Lin et al. (1999)
XP003612186 TLP 64% Medicago truncatula Unpublished
BoTLP9 ABV89616 PR TLP 88% B. rapa Ahmed et al. (2013)
NP177708 PR TLP 84% A. thaliana Theologis et al. (2000)
BoTLP10 ABV89616 PR TLP 98% B. rapa Ahmed et al. (2013)
NP177708 PR TLP 85% A. thaliana Theologis et al. (2000)
BoTLP11 NP173365 PR TLP 67% A. thaliana Theologis et al. (2000)
BAD90814 TLP 55% C. japonica Futamura et al. (2006)
BoTLP12 NP197850 PR TLP 79% A. thaliana Unpublished
BAB11214 TLP 91% A. thaliana Sato et al. (1998)

z)Analyzed using BLAST from NCBI, http://www.ncbi.nlm.nih.gov/BLAST/.

y)PR TLP- Pathogenesis related thaumatin-like protein.

Table 1 Primer information of 12 thaumatin-like genes of B. oleracea for RT and Real time PCR.
Table 2 Overall analysis of thaumatin-like genes collected from the Brassica oleracea database.z)

Brassica oleracea database (http://119.97.203.210/bolbase/blast/blast.html).

Table 3 Homology analysis of 12 thaumatin-like genes of Brassica oleracea z)

Analyzed using BLAST from NCBI, http://www.ncbi.nlm.nih.gov/BLAST/.

PR TLP- Pathogenesis related thaumatin-like protein.

Table 1

Table 2

Table 3

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