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Tau class glutathione S-transferases candidates from Brassica rapa : Genetic mapping and sequence analysis

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Glutathione S-transferases (GSTs) have medically and agriculturally important roles in plants such as herbicide detoxification, responses to biotic and abiotic stress and catalytic reaction of anticancer compounds. The fully sequenced Arabidopsis
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  Tau Class Glutathione S-transferases Candidates from  Brassica rapa :Genetic Mapping and Sequence Analysis Tae-Ho Park  1,2 , Mina Jin 1 , Sang-Choon Lee 1,3 , Joon Ki Hong 1 , Young-Joo Seol 1 ,Jeong-Hwan Mun 1 , and Beom-Seok Park  1 * 1  Department of Agricultural Biotechnology, National Academy of Agricultural Science, Suwon 441-707, Korea 2  Department of Horticultural Science, Daegu University, Gyeongsan 712-714, Korea 3  Department of Life Science, Sogang University, Seoul 121-742, Korea *Corresponding author: pbeom@korea.kr  Received July 13, 2010 / Accepted February 23, 2010  Korean Society for Horticultural Science and Springer 2011 Abstract. Glutathione S-transferases ( GSTs ) have medically and agriculturally important roles in plants such as herbicide detoxification, responses to biotic and abiotic stress and catalytic reaction of anticancer compounds. The fully sequenced  Arabidopsis thaliana  and rice ( Oryza sativa ) genome revealed identification of 52 and 61 members of GSTs , and 28 and 39 of those belong to the Tau class of GSTs  ( GSTUs ), respectively. Based on the sequences of  AtGSTUs , 14 BAC clones derived from  Brassica rapa  that contain similar sequences of  AtGSTUs  were identified and 17 unique sequences of open reading frames were detected in the 14 BAC clones using blast search and sequence alignment. Those sequences were designated the Tau class candidate of GST   derived from  B. rapa  (  BrGSTUs ) and it is confirmed that  BrGSTUs  are also clustered in tandem as it has been known to be common feature in plant. They were mapped on BrR5, BrR7, BrR8, BrR9, or BrR10, and their nucleotide and amino acid sequences were highly similar to those of  AtGSTUs . In addition, in silico  analysis of  BrGSTUs  was  performed using Korea Brassica Genome Project 24 K (KBGP-24K) oligochip and microarray database for cold, salt, and drought stresses. The results of this study will facilitate further researches for breeding of Chinese cabbage containing medically and agriculturally important traits.  Additional key words :  arabidopsis, chinese cabbage, comparative, in silico  analysis, microarray, phylogenetic analysis Hort. Environ. Biotechnol. 52(3):284-291. 2011.DOI 10.1007/s13580-011-0141-5 Research Report  Glutathione S-transferases ( GSTs ) are multifunctional  proteins ubiquitously distributed in highly diverse aerobic organism, from bacteria to human. These proteins have fun-damental roles in the cellular detoxification of a wide range of exogenous and endogenous compounds. In bacteria, they are involved in the reactions of antibiotic resistance and degradation pathways of chemicals and in mammals, they detoxify chemical carcinogens and function as chemothe-rapeutic agents (Frova, 2003, 2006). GSTs  have been grouped into six classes, tau (U), phi (F), theta (T), lambda (L), zeta (Z) and DHAR, based on seque-nce similarity, gene organization, and active site residues in the proteins (Dixon et al., 2002a; Frova, 2003). GSTs  from the phi ( GSTF  ) and tau ( GSTU  ) classes are unique to plants and functions of GSTs  in plants are associated with a wide range of biotic and abiotic stresses including herbicide, organic pollutants, natural toxins, and diseases with regards to detoxification and environmental safety (Frova, 2003; Hatton et al., 1996; Neuefeind et al., 1997c; Nutricati et al., 2006). Especially GSTs  are enzymes catalyzing the conjuga-tional reaction on glucosinolate metabolic pathways with glutathione and isothiocyanate including sulforaphane in crucifer crops (Rea, 1999). Sulforaphane is one of the major functional ITCs which is known as anticancer compound and derived from the 4-methylsulfinybutyl glucosinolate that accumulates in Brassicaceae crops, especially broccoli (  Brassica oleracea ) (Ambrosone et al., 2004; Gasper et al., 2005).As identified in the fully sequenced  A. thaliana  and rice ( Oryza sativa ) genome, Tau class GSTs  ( GSTUs ) is the  biggest group of GSTs  in plants. 28 of 52  Arabidopsis GSTs (  AtGSTs ) and 39 of 61 rice GSTs  ( OsGSTs ) belong to Tau class of GSTs  (Dixon et al., 2002a, 2002b; Frova, 2003; Wagner et al., 2002). They mostly show the non-random distribution. In  Arabidopsis  genome, 18 and 7 out of 28  AtGSTUs  are located on chromosome 1 and 2, respectively  Hort. Environ. Biotechnol. 52(3):284-291. 2011. 285 and especially seven  AtGSTUs  are clustered in tandem in a 14 kb segment of chromosome 2 (Lin et al., 1999). In rice genome, 29 out of 39 OsGSTUs  map to chromosome 10 and 24 were clustered within 240 kb (Yuan et al., 2002). In a previous study, we reported genetic mapping and sequence analysis of Phi class GSTs  from  Brassica rapa (  BrGSTFs ) (Park et al., 2008) using database of sequenced BAC and ESTs clones, Korea Brassica Genome Project 24 K oligochip (KBGP-24K) and microarray database (http:// www.brassica-rapa.org/BGP/NC_brgp.jsp; Lee et al., 2008) generated as a part of Brassica Genome Sequencing Project. In this study, as an extended study, we tried specifically to identify Tau class GSTs  from Chinese cabbage (  B. rapa )(  BrGSTUs ) using our database. We mapped the identified  BrGSTUs  and compared the sequences of  BrGSTUs  with those of  AtGSTUs . In addition, in silico  analysis of gene expression was performed using KBGP-24K and microarray database.            First of all, sequences of Tau class of  A. thaliana  (  AtGSTUs )were collected from publically available database, The  Ara-bidopsis  Information Resource (TAIR) database (http://www. arabidopsis.org) and NCBI (http://www.ncbi.nlm.nih.gov/). Based on the sequences of  AtGSTUs , searches for BAC clones containing Tau class of GSTs  from  B. rapa  (  BrGSTUs )were performed. The BAC clones srcinating from three different large-insert BAC libraries, KBrH (  Hin dIII), KBrB (  Bam HI) and KBrS ( Sau 3AI) were previously sequenced for full sequencing of  B. rapa  ssp. pekinensis cv. Chiifu (http: //www.brassica-rapa.org/BGP/; Park et al., 2005; Yang et al., 2005) and registered at NCBI. ‘tblastx’ that searches translated nucleotide database (the sequenced BAC clones) using a translated nucleotide query (  AtGSTUs ) was performed at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and our own database.      The map positions of the selected BAC clones harboring  BrGSTUs  were determined using two mapping populations JWF3p (Kim et al., 2006) and VCS3-DH (Jin et al., unpu-lished). They were compared with those of  AtGSTUs  physi-cally defined on  A. thaliana  chromosomes at TAIR database (http://www.Arabidopsis.org).      The web-based gene prediction programs, GeneMark (http://opal.biology.gatech.edu/GeneMark/; Lukashin and Borodovsky, 1998) and Genescan (http://genes.mit.edu/ GENSCAN.html; Burge and Karlin, 1997) were used to analyze the selected BAC clones expected to contain the sequences of the candidate  BrGSTUs . According to the results, the sequences of  BrGSTFs  were dissected from the BAC clones and compared with those of  AtGSTFs . AlignX in Vector NTI suite 9 (Invitrogen, Carlsbad) and a web-  based software, PipMaker (Schwartz et al., 2000) were used to investigate sequence similarity and sequence colinearity, respectively.          BrGSTUs  were analyzed to investigate their expression in  silico  using KBGP-24 K and microarray database for cold, salt and drought stresses (http://www.brassica-rapa.org/BGP /NC_brgp.jsp). Lee et al. (2008) reported 23,939 unigenes  presented in KBGP-24 K and these data were used to blast the  Arabidopsis  sequences and all unigenes were assigned to the loci of  Arabidopsis . Therefore  BrGSTUs  and candidate unigenes similar to the loci of  AtGSTUs  were selected. These selected unigenes were analyzed in the microarray database generated for cold, salt and drought stresses. The expression patterns of the candidate  BrGSTUs  were gener-ated using a MeV software (http://www.tm2.org/mev. html).           To select BAC clones harboring the candidate  BrGSTUs ,28  AtGSTUs  (from  AtGSTU1  to  AtGSTU28 ) were collected from TAIR database (http://www.Arabidopsis.org) (Table 1). The sequences of  AtGSTUs  were blasted to the seque-nced BAC clones derived from  B. rapa  ssp. pekinensis cv. Chiifu using ‘tblastx’ at NCBI (http://blast.ncbi.nlm.nih. gov/Blast.cgi) and our own database. 14 BAC clones conta-ining full length of open reading frames (ORFs) which were highly similar with any of  AtGSTUs ’ sequences. Their accession numbers registered in the NCBI database (http:// www.ncbi.nlm.nih.gov/) were shown in Table 2. All selected BAC clones were annotated using GeneMark (http://opal.  biology.gatech.edu/GeneMark/; Lukashin and Borodovsky, 1998) and Genescan (http://genes.mit.edu/GENSCAN.html; Burge and Karlin, 1997) and 17 predicted  BrGSTU   genes were dissected from the 14 BAC clones for further analysis. Depending on the sequence similarity or identity of the dissected sequences, they were designated as one of  BrGSTUs (Table 2). One, three, two, and one BAC clones contain one copy of the sequences correspondent to  AtGSTU5 ,  AtGSTU13 ,  AtGSTU18 , and  AtGSTU26  , respectively and they were identical within two groups (  AtGSTU13  and  AtGSTU18 ). However, the rest of the selected BAC clones contains different copy number of GSTUs . For instance, the BAC clone, KBrB006F18 consists of four different sequences  Tae-Ho Park, Mina Jin, Sang-Choon Lee, Joon Ki Hong, Young-Joo Seol, Jeong-Hwan Mun, and Beom-Seok Park  286 Table 1. List of the  AtGSTUs  and the number of unigenes selected from B. rapa  database. This table was partially adopted from Wagner et al. (2002).NameAt locus IDNumber of unigenes z Old name y Reference AtGSTU1At2g294900GST19unpublished AtGSTU2At2g294800GST20unpublished AtGSTU3At2g294701GST21unpublished AtGSTU4At2g294602GST22unpublished AtGSTU5At2g294502 AT103-1avan der Kop et al. (1996) AtGSTU1Edwards et al. (2000) AtGSTU6At2g294400GST24unpublished AtGSTU7At2g294202GST25unpublished AtGSTU8At3g092700-unpublished AtGSTU9At5g624800GST14unpublished AtGSTU10At1g745901-unpublished AtGSTU11At1g699302-unpublished AtGSTU12At1g699203-unpublished AtGSTU13At1g271303GST12unpublished AtGSTU14At1g271400GST13unpublished AtGSTU15At1g596700-unpublished AtGSTU16At1g597001-unpublished AtGSTU17At1g103701GST30unpublished AtGSTU18At1g103601GST29unpublished AtGSTU19At1g783801GST8Bianchi et al. (2002) AtGSTU20At1g783701-unpublished AtGSTU21At1g783600-unpublished AtGSTU22At1g783401-unpublished AtGSTU23At1g783200-unpublished AtGSTU24At1g171701-unpublished AtGSTU25At1g171803-unpublished AtGSTU26At1g171900-unpublished AtGSTU27At3g438001-unpublished AtGSTU28At1g536800-unpublished z Number of unigenes detected in microarray database of B. rapa  generated for cold, salt and drought stresses. y  A few names have been changed due to the new  AtGST   nomenclature suggested by Edwards et al. (2000). correspondent to  AtGSTU19 ,  AtGSTU20 ,  AtGSTU22 , and  AtGSTU23 , and then they were designated  BrGSTU19  _c,  BrGSTU20  _c,  BrGSTU22  _c, and  BrGSTU23  _c, respectively. Although two BAC clones (KBrH062L23 and KBrH109O07) selected for  AtGSTU25  contain three and two copies, respectively, they were all different each other. They were, therefore, named as from  BrGSTU25  _c1 to  BrGSTU25  _c5.      From the selected 14 BAC clones, 17  BrGSTUs  were identified as the candidates and genetically positioned on two different mapping populations. Their relative physical  positions are shown in Fig. 1B. They were randomly distrib-uted on five different  B. rapa  linkage groups. Especially five  BrGSTU25  derived from two different BAC clones were mapped on the same region of  B. rapa  linkage group R9. The map positions of all  AtGSTUs  were also indicated on five  Arabidopsis  chromosomes (Fig. 1A).                Phylogenetic tree was constructed with amino acid sequences of 28  AtGSTUs  and 17 candidate  BrGSTUs  using the neighbor-joining method (Saitou and Nei, 1987) in Vector NTI suite 9 (Invitrogen, Carlsbad) as shown in Fig. 2. It was built on a matrix of distances between all pairs of sequences to be analyzed. These distances are related to the degree of divergence between the sequences. The design-  Hort. Environ. Biotechnol. 52(3):284-291. 2011. 287 Table 2. List of the selected BAC clones and candidate BrGSTUs .Name of AtGSTBAC clonesAccession numberCopy numberName of BrGST AtGSTU5KBrS010D18AC2409471BrGSTU5_c AtGSTU11 & AtGSTU12KBrB045C17AC2409323BrGSTU11_c1, BrGSTU12_c1, & BrGSTU12_c2KBrB064F02AC2325113BrGSTU11_c1, BrGSTU12_c1, & BrGSTU12_c2KBrB064L24AC2409333BrGSTU11_c1, BrGSTU12_c1, & BrGSTU12_c2KBrB073K15AC1894591BrGSTU11_c2 AtGSTU13KBrB024J13AC1892741BrGSTU13_cKBrB043N19AC2409311BrGSTU13_cKBrB096H15AC2409341BrGSTU13_c AtGSTU18KBrH103O15AC2409451BrGSTU18_cKBrS004C14AC2325711BrGSTU18_c AtGSTU19, AtGSTU20,  AtGSTU22 & AtGSTU23KBrB006F18AC2324454BrGSTU19_c, BrGSTU20_c, BrGSTU22_c, & BrGSTU23_c AtGSTU25KBrH062L23AC2409443BrGSTU25_c1, BrGSTU25_c4 & BrGSTU25_c5KBrH109O07AC2409462BrGSTU25_c2, & BrGSTU25_c3 AtGSTU26KBrS004B22AC1728871BrGSTU26_c  Fig. 1. Genetic positions of   GSTUs  on  A. thaliana  chromosomes (A) and B. rapa chromosomes (B). The length (Mbp) of each  A. thaliana  chromosome is indicated on the right of the gray bars. The positions of all  AtGSTUs  were adopted from TAIR database (http://www.arabidopsis.org). The positions of cen-tromeres of B. rapa  chromosomes are represented by the black boxes. Fig. 2. Phylogenetic tree of GSTUs  identified in  A. thaliana  and B. rapa . The calculated distance values in parentheses following the name of GSTUs  are displayed. ation of  BrGSTUs  (Table 2) resulted from this phylogenetic tree. According to the results of the phylogenetic tree, nine groups of GSTUs  were further investigated separately. The amino acid and nucleotide sequences of GSTUs  in nine groups were aligned (results not shown) and pairwise amino acid and nucleotide sequence similarity in each group were examined (Fig. 3) using AlignX in Vector NTI suite 9 (Invitrogen, Carlsbad). The similarities of amino acid seq-uences and nucleotide sequences of all  At/BrGSTUs  in each  Tae-Ho Park, Mina Jin, Sang-Choon Lee, Joon Ki Hong, Young-Joo Seol, Jeong-Hwan Mun, and Beom-Seok Park  288       Fig. 3. Pairwise amino acid and nucleotide sequence similarities in nine groups of   GSTUs  containing different BrGSTUs . A: BrGSTU5  ,B: BrGSTU11 , C: BrGSTU12  , D: BrGSTU13 , E: BrGSTU18  , F: BrGSTU19 , BrGSTU20   and BrGSTU22  , G: BrGSTU23 , H: BrGSTU25  ,and I: BrGSTU26  . group were all high and it was confirmed that the candidate  BrGSTUs  were the most likely to be close to  AtGSTU5 ,  AtGSTU11 ,  AtGSTU12 ,  AtGSTU13 ,  AtGSTU18 ,  AtGSTU19 ,  AtGSTU20 ,  AtGSTU22 ,  AtGSTU23 ,  AtGSTU25 , and  AtGS-TU26   in each group. The group which showed the highest similarities at both amino acid and nucleotide sequence levels between  AtGSTU   and  BrGSTU   was GSTU11  ranging from 92% to 94% and from 90% to 91%, respectively (Fig. 3B). The group which showed the lowest similarities between  AtGSTU   and  BrGSTU   at amino acid sequence level was GSTU12  ranging from 70% to 71%, but GSTU25  for nucle-otide sequence level ranging from 62% to 73% (Figs. 3C and 3H). In the biggest group being comprised of  AtGSTU25 and  BrGSTU25  _c1-c5, it ranged from 68% to 85% for amino acid sequences between  AtGSTU25  and  BrGSTU25 (Fig. 3H). Sequence similarity and colinearity within nine groups were confirmed using PipMaker (results not shown). Similarity within  BrGSTUs  ranged from 65% to 100% at the amino acid sequence level and from 73% to 99% at the nucleotide sequence level.         KBGP-24 K oligochip consisting of 23,929 unigenes and microarray database for cold, salt, and drought stresses (http://www.brassica-rapa.org/BGP/NC_brgp.jsp) were used for digital northern analysis of  BrGSTUs . All the sequences of unigenes were assigned to the locus of  A. thaliana  based on the best matches at the sequence blast. Of 23,929 unigenes on the KBGP-24 K oligochip, 27 were detected to  be similar to one of 17  AtGSTUs  (Table 1).The selected 27 unigenes were investigated in the results of microarray analyses of  B. rapa  plants treated with abiotic stresses including cold, salt and drought. Their expression  patterns are presented in Fig. 4. The color scale indicated the fold-change of genes converted from perfect match (PM) values of the six probes which were designed in the sense direction (5’-UTR to 3’-UTR) (Lee et al., 2008). Seventeen of the selected 27 unigenes, BRAS0001S00000113 (only last five digits used from here) and 14692 (  AtGSTU5 ), 06370 and 14791 (  AtGSTU11 ), 01944, 15230, and 25245 (  AtGSTU12 ), 00582, 00583 and 17586 (  AtGSTU13 ), 06819 (  AtGSTU18 ), 00122 (  AtGSTU19 ), 14265 (  AtGSTU20 ), 12595 (  AtGSTU22 ), and 01077, 03840, and 14226 (  AtGSTU25 )were compared with those of candidates  BrGSTU5 ,  BrGSTU11 ,  BrGSTU12 ,  BrGSTU13 ,  BrGSTU18 ,  BrGSTU19 ,  BrGSTU20 ,  BrGSTU22 , and  BrGSTU25  because they were selected and comparable in both  B. rapa  BAC clones and unigenes. 00113 (  AtGSTU5 ), 06370 (  AtGSTU11 ), 15230 (  AtGSTU12 ),
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