Genomic survey, characterization and expression profile analysis of the peptide transporter family in rice (Oryza sativa L.)
© Zhao et al; licensee BioMed Central Ltd. 2010
Received: 22 January 2010
Accepted: 20 May 2010
Published: 20 May 2010
Peptide transporter (PTR) family whose member can transport di-/tripeptides and nitrate is important for plant growth and development. Although the rice (Oryza sativa L.) genome has been sequenced for a few years, a genomic survey, characterization and expression profile analysis of the PTR family in this species has not been reported.
In this study, we report a comprehensive identification, characterization, phylogenetic and evolutionary analysis of 84 PTR family members in rice (OsPTR) as well as their whole-life expression patterns. Chromosomal distribution and sequence analysis indicate that nearly 70% of OsPTR members are involved in the tandem and segmental duplication events. It suggests that genome duplication might be a major mechanism for expansion of this family. Highly conserved motifs were identified in most of the OsPTR members. Meanwhile, expression profile of OsPTR genes has been analyzed by using Affymetrix rice microarray and real-time PCR in two elite hybrid rice parents, Minghui 63 and Zhenshan 97. Seven genes are found to exhibit either preferential or tissue-specific expression during different development stages of rice. Under phytohormone (NAA, GA3 and KT) and light/dark treatments, 14 and 17 OsPTR genes are differentially expressed respectively. Ka/Ks analysis of the paralogous OsPTR genes indicates that purifying selection plays an important role in function maintenance of this family.
These investigations add to our understanding of the importance of OsPTR family members and provide useful reference for selecting candidate genes for functional validation studies of this family in rice.
Nutrient transport is essential to life and occurs in both prokaryotes and eukaryotes. It is well established that peptide transporters play an important role in the nutrition of bacteria, yeasts and animals . However, the role of transporters of small peptides (2-6 amino acids) in plants is less well defined . Peptide transporter (PTR) family (TC 2.A.17), also called the proton-dependent oligopeptide transport (POT) family [3, 4] is a family consists of di-/tripeptides transporters . Besides, some plant PTRs acted as nitrate transporters are termed as the members of Nitrate Transporter 1 family (NRT1, ). PTR proteins in plants show significant sequence homology and contain several transmembrane (TM) regions, with a large hydrophilic loop among TMs. Generally, the identified PTR members in plants fall into three types based on the nature of their substrates: di-/tripeptides transporter, nitrate transporter and other substrates transporter .
AtPTR2 (At2g02040) was the first identified di-/tripeptides transporter in Arabidopsis [7–10] and had high mRNA expression levels in 3-d-germinating seed, root and young leaf . The antisense plants of AtPTR2 exhibited delayed flowering time and arrested seed development [9, 11]. AtPTR1 (At3g54140) transported di-/tripeptides with low selectivity as well as substrates lacking a peptide bond. AtPTR1 was expressed in the vascular tissue throughout the plant, indicative of a role in long-distance transport . AtPTR3 (At5g46050) was a salt stress and mechanical wounding inducible gene, JA and SA were both involved in regulation of it [12, 13]. AtPTR5 (At5g01180) which mediated high-affinity transport of dipeptides was most likely supplying peptides to maturating pollen, developing ovules and seeds. Overexpression of it resulted in enhanced shoot growth and increased N content . The barley peptide transporter gene, HvPTR1, had seed-specific expression. Transport activity of HvPTR1 was regulated by phosphorylation in response to rising levels of amino acids in the germinating grain [15–17]. Functional di-/tripeptides transporters also have been reported in Nepenthes (NaNTR1, ), faba bean (VfPTR1, ) and Hakea actites (HaPTR4, ).
Although PTR is the abbreviation of peptide transporter, many cloned PTR members in plants are nitrate transporters, especially in Arabidopsis. AtNRT1.1 (At1g12110, CHL1), a dual-affinity nitrate transporter, was regulated by phosphorylation and involved in nascent organ development [21–26]. AtNRT1.2 (At1g69850, NTL1) encoded a low-affinity nitrate transporter. RNAi plants of it exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities . The characterization of AtNRT1.4 (At2g26690) revealed the special role of petiole in nitrate homeostasis . AtNRT1.5 (At1g32450) and AtNRT1.6 (At1g27080) were both plasma membrane proteins. AtNRT1.5 was expressed in root pericycle cells close to the xylem and participated in root xylem loading of nitrate  while AtNRT1.6 was only expressed in reproductive tissue and involved in delivering nitrate from maternal tissue to the developing embryo . CsNitr1, a nitrite transporter in Cucumis sativus, had two isoforms: CsNitr1-S and CsNitr1-L. In contrast to that CsNitr1-S enhanced the efflux of nitrite from the cell, CsNitr1-L might load cytosolic nitrite into chloroplast stroma .
Amino acids and other substrates can also be transported by some PTRs. BnNRT1.2, isolated from Brassica napus could transport both nitrate and histidine . AgDCAT1 localized at the symbiotic interface was a dicarboxylates transporter in Alnus glutinosa and the mRNA of AgDCAT1 was only detected in the nodules .
In rice, only two PTR genes have been functionally verified. OsNRT1 (LOC_Os03g13274), which displayed low-affinity nitrate transport activity was constitutively expressed in the most external layer of the root, epidermis and root hair . SP1 (LOC_Os11g12740) which determined the panicle size had high expression level in the phloem of the branches of young panicles. Phylogenetic analysis implied that SP1 might be a nitrate transporter, however, neither nitrate nor other compounds transport activity could be obtained from it .
As the main staple food for a large segment of the world population, rice also serves as a model plant for monocotyledon species [36, 37]. Nutrient transport is critical for plant growth and development, in which PTR family members may play important roles. This work focused on a comprehensive identification, characterization, phylogenetic and evolutionary analysis of all the PTR family members in rice (OsPTR) as well as their whole-life expression profiling. Moreover, it has been demonstrated that the expression of some OsPTR genes is regulated phytohormone (NAA, KT and GA3) and light/dark treatments.
Collection and identification of PTR genes in rice
Domain search (PF00854) of MSU Rice Genome Annotation Project (MSU RGAP) database released 125 sequences, out of which 91 were unique. Keywords "PTR", "peptide transporter" and "proton-dependent oligopeptide transport" searching identified 58, 85 and 10 genes respectively. BLASTP and TBLASTN searches against rice genome in three databases: MSU RGAP, NCBI and KOME released 95, 85 and 116 sequences respectively. By removal of the same sequences and different transcripts of the same gene, we initially identified 93 putative PTR genes in rice. Protein sequences of these genes were subjected to Pfam and SMART searches for the presence of PTR domains. As a result, seven genes whose protein contained only partial PTR domain and two genes annotated as retrotransposons were excluded from further analysis. Taken together, a total of 84 genes were predicted to encode PTR proteins in rice. Eighty-three genes had corresponding locus IDs in MSU RGAP database and one gene (Os01g0960900) was only found in NCBI. For convenience, all the "LOC_" prefix of MSU RGAP locus IDs were omitted representing OsPTR genes or proteins for further analysis. Meanwhile, similar methods were used and 53 PTR family members in Arabidopsis (AtPTR) were identified. The detailed information about the full-length cDNA, transcript, chromosomal position, BAC accession, gene structure for each OsPTR gene and characteristics of corresponding proteins can be found in Additional file 1. Protein structures predicted by SMART are shown in Additional file 2. The putative transmembrane (TM) regions in each OsPTR protein are listed in Additional file 3.
Chromosomal localization and gene duplication
Analysis of the MSU RGAP segmental duplication database revealed that 21 OsPTR genes (11 pairs) could be assigned to rice segmental duplication blocks (Figure 1). Os02g47090 participated in two segmental duplications with Os04g50940 and Os10g42870 respectively. The overall identities of the protein sequences of these genes ranged from 37.6% to 76.1%. A total of 44 genes (14 clusters, nine genes also involved in segmental duplication) were considered as tandem duplications according to the criterion adopted in our analysis, including the largest cluster of nine genes on chromosome 1, two four-gene clusters on chromosome 10 and 5, five three-gene clusters on chromosome 3, 4, 6 and 11, six two-gene clusters on chromosome 1, 5, 6, 10 and 12 (Figure 1). The protein homology of these genes varied from 22.9% to 89.5%. Interestingly, all the tandemly duplicated genes in the same cluster had the same direction of transcription except Os06g13200 and Os06g13210. This might suggest the conserved behavior of tandem duplications in this family. Of the 84 OsPTR genes, 66.7% (56 of 84) involved in the duplication events. Moreover, most of the duplicated genes had relative close phylogenetic relationship (see below). Therefore, segmental and large-scale tandem duplication events appeared to have exclusively contributed to the expansion of the OsPTR gene family. Similar manner of member duplication had also been described in some large gene families in rice, for instance, the ankyrin repeat gene family  and the bHLH family .
Phylogenetic analysis and multiple sequence alignment
In order to investigate the relationship between the function and phylogenetic subfamily formation of PTR members as well as identify some orthologous genes, a combined phylogenetic tree with OsPTR and AtPTR proteins was also established (Additional file 4). As a result, similar subfamilies were formed compared to the tree of OsPTR. Each subfamily contained both Arabidopsis and rice PTR members. However, most of the members were clustered in species-specific distinct clades, and only four pairs of orthologs (Os01g01360 and At5g13400, Os01g37590 and At2g26690, Os06g15370 and At1g68570, Os05g27010 and At2g40460) could be figured out. This result indicated that the main characteristics of PTR family in rice and Arabidopsis were formed before the split of monocotyledonous and dicotyledonous plants and then evolved separately in a species-specific manner. Moreover, the difference in the total number of OsPTR and AtPTR was mainly due to the variation in the number of subfamily I genes, which were 30 in rice and eight in Arabidopsis. Of the 30 subfamily I PTRs in rice, 21 were tandemly or segmentally duplicated genes.
Expression profiling of OsPTRgenes in whole-life cycle of rice
Based on hierarchical clustering, expression pattern of OsPTR genes could be divided into five major groups. Group 1 consisted of 25 genes which showed high expression levels in all the tissues expect six genes that showed relatively low expression in certain tissues (Os03g60850 in Sta, Os03g04570 in Ste1, P1, Sta and E3, Os06g15370 in FL1, FL2 and Ste1, Os10g02240 in P1-P3, Os10g33170 in Se, Sta, E1 and E3 and Os12g44100 in P1-P3). Conversely, group 2 contained 10 genes that showed low expression level in the whole life cycle. Group 3 was comprised of 16 genes that showed relatively high expression level in both vegetative tissues and hull. All these genes had low expression level in early panicle and endosperm stages. Group 4 comprised of 16 genes with medium expression level in all the tissues except that Os04g56560 had high expression in root, Os01g65120 in E2, Os11g23890 in E1-E2 and Os01g55600 in E1-E3. The rest 14 genes belonged to group 5 although these genes had different expression patterns. For example, Os04g41400 had high expression signal in root and stamen, Os04g59480 showed high expression in FL2, Os11g12740 had high expression in panicle and stem. Interestingly, Os01g65110, Os01g55610, Os11g17970, Os10g42900 and Os06g21900 all showed relatively high expression in endosperms. The expression patterns of most OsPTR genes in Zhenshan 97 (ZS97) were similar to that in MH63 and the corresponding hierarchical cluster was shown in Additional file 6.
The hierarchical cluster of the AtPTR genes in 24 development stages released the expression profile of 51 genes except At1g72125, At3g45660 and At3g54450 (Additional file 7). Obviously, the expression patterns of AtPTR genes fell into the similar groups as OsPTR genes. Some tissue-specific expression AtPTR genes could also be identified, for example, At1g27040, At4g21680 and At3g25260 in seed and siliques, At3g45710 and At3g45700 in root. Most of the AtPTR genes had relatively low expression level in mature pollen except At1g62200, At5g28470 and At5g01180. These genes were most likely supplying nutrition to maturating pollen. Some of the orthologous OsPTR and AtPTR genes showed the similar expression pattern such as Os06g15370 and At1g68570, Os05g27010 and At2g40460. They both had high expression level in all the tissues. However, no more apparent relationship between the expression pattern and sequence similarities of OsPTR and AtPTR members could be pointed out.
Expression regulation of OsPTRgenes under phytohormone and light/dark treatments
Ka/Ks analysis and express pattern divergence of paralogous OsPTRgenes involved in duplication
Ka/Ks and divergence analysis of paralogous OsPTR genes involved in duplication
Di-/tripeptides and nitrate transport is important for various aspects of plant growth and development, in which peptide transporter family members may play important roles. Considering the potential functional diversity of PTR members associated with nutrient transport and only a few PTR family members have been described in higher plants, it was timely and quite relevant for us to characterize the rice PTR family. Combination of the whole genome sequence data, sequences alignment and high-throughput expression analysis undertaken in this investigation provides an insight into all the 84 members of PTR family in rice.
Duplication played an important role in the expansion of the OsPTRfamily
It is well known that gene duplication is important for gene family evolution and diversity, which can occur via three major mechanisms: segmental duplication, tandem duplication and retroposition . Due to repetitive plant genome duplications, certain plant genomes are likely to harbor relatively large members of a gene family, as compared to animal genomes . The PTR families in rice and Arabidopsis are very much in line with the statement and showed increased family members in order to adapt for specific functions. The number of 84 OsPTR and 53 AtPTR members is far greater as compared to the low number of six in human, four in C. elegans, three in Drosophila and two in yeast . It may indicate the importance of these transporters in plant growth and metabolism. Nearly 70% of the 84 OsPTR genes are duplicated genes, 21 genes (25%) are found to be involved in the segmental duplication while 44 (53.4%) participated in tandem duplication. Our analysis also indicated that in subfamily I, duplication events were largely responsible for the number difference of the member between OsPTR and AtPTR. It demonstrates that the OsPTR family may undergo extensive and rapid genomic changes which can maintain relative large number of member and lead to expression divergences between duplicate genes, further contributing to the establishment of novel gene functions .
Phylogenetic relationship and functional diversity among the OsPTR members
The phylogenetic analysis reveals that the OsPTR family can be further divided into five subfamilies. The combined phylogenetic tree of OsPTR and AtPTR showed similar subfamily formation whereas most PTR members were clustered in species-specific clades and only four pairs of orthologs could be identified. It suggests that the members of the PTR family have diverged from each other primarily due to speciation and then undergone significant changes since the monocot/dicot split.
However, highly conserved motifs have been identified in most members of AtPTR and OsPTR. We further adopted the sequence alignment including the PTR members in plant, animal, human and prokaryotic organisms. It was surprising to find that the three motifs could be found in most eukaryotic PTR members but changed a lot or lost in the prokaryotic PTR members, especially motif 2 and motif 3. Moreover, some members such as Os01g65120, Os04g50930, Os12g13790 and At2g38100 which had far phylogenetic relationship from other PTR members, had great amino acid variation in motif 2 and motif 3. We inferred that motif 2 and motif 3 might be important characteristic logos of the eukaryotic PTR members and played considerable roles in the function formation of the PTR family. Further research may employ the point mutations introduced into these motifs, which should cause a loss of function of the corresponding protein. This experimentation could indicate the essentiality of these motifs for the formation of the transporter pore and the interaction with the substrates.
The sequence similarity among the OsPTR members seemed to show little relationship with their putative transporter type classification. In addition, we found that gene structures, intron phases of OsPTR genes and distribution of TM regions in the proteins had no relationship with their phylogenetic subfamily and transporter type classifications. As most OsPTR proteins had PTR as their only recognizable domain (See Additional file 2), we adopted the MEME method , in order to identify some new motifs or domains. However, the conserved motifs investigated by MEME were all TM regions (data not shown). We infer that the substrate selection of OsPTR proteins may be decided by their three dimensional structures. The determination of the di-/tripeptide, nitrate and other type transporters in this family by mutagenesis or crystal structure studies should solve the puzzle that di-/tripeptides, nitrate and other substrates of PTR proteins are very different in structure but they share the same transporter family - PTR .
Expression pattern divergence of the OsPTRgenes
Microarray provides a rich resource for investigating the evolution of gene expression. The temporal and spatial expression patterns of OsPTR genes are useful information for establishing their putative functions. Our microarray analysis indicated that the expression patterns of the 81 OsPTR genes could be classified into five groups. We also identified some preferential or tissue-specific expression OsPTR genes. Of the 81 genes analyzed in this study, only seven were found to exhibit either preferential or tissue-specific expression during different development stages of MH63 and ZS97. Interestingly, these genes were most preferentially or specifically expressed in root and stamen. Os02g47090, Os04g50940 and Os04g50950 were preferentially expressed in root. Moreover, these three genes were in the same phylogenetic clade that belonged to the group three of subfamily I. In Arabidopsis, the expression of AtNRT1.1 was found concentrated in the tips of primary and lateral roots . AtNRT1.2 was primarily expressed in root hairs and the epidermis in both young and mature regions of roots . OsNRT1 was constitutively expressed in the most external layer of the root, epidermis and root hair . Our data showed that OsNRT1 had high expression levels in all the tissues especially in root. Os03g13240 and Os03g13250 were the paralogs of OsNRT1. Moreover, these three genes formed a tandem duplication cluster. Os03g13240 and Os03g13250 showed similar expression patterns with OsNRT1 and Os03g13250 had the highest expression level in root. It can be inferred that these root-preferentially or -specifically expressed OsPTR genes might be nitrate transporters and play important roles in root formation and development. AtNRT1.4 was expressed in the leaf petiole and participated in regulating leaf nitrate homeostasis . The ortholog of AtNRT1.4 in rice was Os01g37590 which continuously expressed in vegetative tissues with high level in leaves. Expression of Os04g50940 was specific in flag leaf of 14-day after flowering both in MH63 and ZS97. Therefore, we infer that Os01g37590 and Os04g50940 might be participated in the development of leaf as well as the nutrient transporting in and/or out of leaf.
In addition, we also identified three genes (Os01g65110, Os04g41410 and Os12g44100) that were preferentially expressed in stamen and one gene (Os01g55600) in endosperm. One interesting phenomenon in our dendrogram was that three-quarter of the OsPTR genes had significant low expression level in panicles of different development stages. SP1 whose transcription accumulated in panicle was one of the exceptions. SP1 was highly expressed in the phloem of the branches of young panicles, which was consistent with the predicted function of SP1 and the phenotype of sp1 mutant . It can be demonstrated that some PTR members might also play roles in nutrient transport in reproductive organ development.
Potential involvement of OsPTR members in phytohormone and light/dark regulations
Little attention has been paid on the functions of PTR members in phytohormone and light/dark regulations. Until now, AtNRT1.1 was the only reported PTR gene that was involved in these regulations. AtNRT1.1 was regulated by auxin in both shoots and roots and involved in light-induced stomatal opening [47, 48]. However, our microarray data showed that the expression patterns of more than one-third OsPTR genes changed significantly under phytohormone and light/dark treatments. Fourteen OsPTR genes are regulated by at least one phytohormone and 17 OsPTR genes are involved in the regulation of light/dark treatment in MH63 and ZS97. From these results, it can be speculated that these differentially expressed OsPTR genes may play an important role in the transport of regulatory molecules (e.g. hormone-peptide conjugates) and other metabolites induced by regulation of phytohormone and light/dark treatments. For example, IAA is an important plant growth phytohormone and 95% of the IAA pool is sequestered in an inactivated conjugated form, e.g. IAA can be conjugated to single amino acids, peptides or proteins via amide bonds. These conjugates may be involved in regulating phytohormone activity in response to plant growth and development, as well as facilitating hormone transport . It seems that the proteins of those OsPTR genes regulated by NAA might have IAA conjugates transport activity.
Fate of the paralogous OsPTRgenes after duplication
From the Ka/Ks analysis of the paralogous OsPTR genes, it is obvious that purifying selection plays an important role in function maintenance of this family. However, comparison of the expression data implied that after duplication, the paralogous pairs may have been evolved in different hypotheses to gain functional divergence. Expression data shows that some of these duplicated genes have undergone subfunctionalization and neofunctionalization, which contribute to the possibility of maintenance and divergence of a paralogous set of genes. Some paralogs have adopted nonfunctionalization which contributes to the likelihood of loss of one member in a pair of paralogous genes. However, sufficient experimentation is required to prove these hypotheses.
In conclusion, the results of this study display the genomic framework, characterization, conserved motifs, duplication manner and evolutionary analysis of the 84 OsPTR members, along with their expression profiles during whole-life stages as well as under phytohormone and light/dark treatments. These data will provide the basis for understanding the evolutionary history of PTR members and their roles in rice growth and development. The findings in our work would be useful in selecting candidate genes for functional validation studies of PTR members in rice. However, future research by adopting RNAi/overexpression strategies or insertion mutagenesis is required to elucidate the precise role of individual OsPTR gene.
Database screening and mining
Three methods were used to identify all putative PTR members in rice. First, domain search was executed on website of MSU RGAP http://rice.plantbiology.msu.edu/domain_search.shtml. Second, key words "PTR", "peptide transporter" and "proton-dependent oligopeptide transport" were used as queries to search against the annotation database of rice genome (Release 6.1 of MSU RGAP). Third, BLASTP and TBLASTN searches with the conserved sequence of PTR download from Pfam http://pfam.sanger.ac.uk/ as query were carried out in three databases: MSU RGAP http://rice.plantbiology.msu.edu/, National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and the Knowledge-Based Oryza Molecular Biological Encyclopedia (KOME, http://cdna01.dna.affrc.go.jp/cDNA). The parameters of blast searches were set as follows: max target sequences-500 and expect value less than 10. The Pfam search (E value < 1.0) and SMART http://smart.embl-heidelberg.de/ search (normal mode, including outlier homologues and homologues of known structure, Pfam domains, signal peptides, internal repeats and intrinsic protein disorder) were used to confirm the presence of PTR domain in each predicted protein. Information about the transcript, chromosomal position, full-length cDNA, BAC accession, gene structure for each gene and characteristics of proteins were obtained from MSU RGAP. The structure of each OsPTR protein was predicted in SMART. To predict the putative TM regions in each OsPTR protein, the TMHMM Server v2.0 http://www.cbs.dtu.dk/services/TMHMM/ was applied with default settings.
Chromosomal localization and gene duplication
The physical positions of OsPTR genes were used to map these genes onto the corresponding rice chromosomes. The distribution of OsPTR genes on chromosomes was drawn by using the MapChart software  and modified manually with annotation. The presence of OsPTR genes on duplicated chromosomal segments was investigated by searching the segmental genome duplication of rice at MSU RGAP http://rice.plantbiology.msu.edu/segmental_dup/index.shtml with the maximum length distance permitted between collinear gene pairs of 500 kb. Gene distributed nearby and separated by five or fewer genes were considered to be tandem duplicates.
Phylogenetic analysis and sequence alignment
ClustalX version 1.83  was used to perform the multiple sequence alignment and generate the unrooted phylogenetic tree based on the protein sequences of OsPTR members with neighbor-joining method . Bootstrap analysis was performed using 1000 replicates. The phylogenetic tree was displayed and annotated using MEGA software version 4 . The combined tree with OsPTR and AtPTR proteins was generated using the same method. The multiple sequence alignment generated by ClustalX was modified manually and the conserved sequences were annotated according to the results of SMART and Pfam searches. The sequence alignment of the conserved motifs was submitted to the hmmbuild program of Mobyle  to build a profile HMM. Then the profile HMM was submitted into LogoMat-M  to draw the HMM logo.
Rice materials and treatments
RNA samples used for microarray hybridization were obtained from two elite hybrid rice parents MH63 and ZS97 growing in normal field conditions at different developmental stages. For phytohormone treatments, seedlings at trefoil stage of MH63 and ZS97 were both treated with solutions of 0.1 mM NAA, GA3 and KT respectively. Samples were collected at the time points of 5, 15, 30 and 60 minutes after treatment and the samples under the same phytohormone treatment of different time points were mixed together. For light/dark treatments, seedlings at the plumule and radicle stages of MH63 and ZS97 were placed under 48 hours continuous light or darkness (when plumule was about 1-2 cm and radicle was about 2-3 cm in length) after emergence and harvested at the assigned time respectively.
Microarray expression data analysis
Expression profile data of OsPTR genes was extracted from CREP database http://crep.ncpgr.cn of our lab that composed of hybridization of RNA samples from 39 tissues/organs covering the whole life cycle of rice with the Affymetrix rice microarray. The microarray data have been submitted under the accession number GSE19024  into the NCBI Gene Expression Omnibus (GEO). Thirteen vegetative and 11 reproductive samples of different developmental stages that could cover the whole life cycle of rice were used for OsPTR expression profile analysis in this study. The detailed information of samples used in analysis is listed in Additional file 9.
Probe sets with the entire set of 11 probes (8-10 in some cases) present on the array with 100% identity over the corresponding genes were considered to be significant. Wherever more than one probe set was available for one gene, the probe set located on the 3' end was given preference. After normalization and variance stabilization, the average signal value of replicates for each probe set was used for analysis. Expression values of each gene were logarithmized in hierarchical cluster analysis and the heatmap were performed using R version 2.9.0 http://www.r-project.org/ with the method of complete linkage. The expression profile data of AtPTR genes was extracted from AtGenExpress (http://www.weigelworld.org/resources/microarray/AtGenExpress, ). Hierarchical cluster analysis and the heatmap drawing were used the same method as OsPTR.
To identify preferentially expressed genes, a student-t test was adopted. A gene was considered as preferential expression gene only if the signal value of the gene in a designated sample was more than two-fold and had a P value less than 0.05 as compared with the other samples. Also, a check of our whole genome expression profile data of rice for different tissues was used to identify tissue-specific expression genes . Under phytohormone and light/dark treatments, genes that were up- or down-regulated more than two-fold with P value less than 0.05 as compared to control were considered as differential expression genes.
Quantitative real-time PCR analysis
Real-time PCR reactions were carried out using the same RNA samples that were used for microarray. The sequences of primers used for real-time PCR are: Os01g65140 (F: AACTGGCTTCTGGCGAAAGAA, R: CGGAAGCAGCTTGACGAATAA), Os05g35650 (F: GGCAGCTGGTTCTCCAACAA, R: CGGCGATCAGCCAGTAGAAG), Os06g13200 (F: GCTCATGTTCGCGGTCATCT, R: TCCATCAACATCCCCTGCTT) and Actin1 (F: TGGCATCTCTCAGCACATTCC, R: TGCACAATGGATGGGTCAGA). First-stand cDNA was synthesized from DNaseI-treated total RNA using Superscript III reverse transcriptase (Invitrogen). Real-time PCR were performed in 25 μl reaction mixture with 1 μl of first-strand cDNA, 12.5 μl 2× SYBR Premix® Ex Taq™ (TaKaRa), 0.5 μl 50× ROX reference dye II and 0.5 μl of each primer. The reactions were carried out using ABI PRISM 7500 system (Applied Biosystems). The following program was used: 95°C for 10 s; 45 cycles of 95°C for 5 s, 60°C for 34 s. Rice Actin1 was used as internal control. The relative expression levels were analyzed as described previously .
Ka and Kscalculations
The paralogous PTR genes in rice were inferred from the phylogenetic tree analysis. Pairwise aligned paralogous protein sequences generated by the ClustalW2 http://www.ebi.ac.uk/Tools/clustalw2/ were used as the alignment guides. PAL2NAL (http://www.bork.embl.de/pal2nal/, ) was used to convert the multiple sequence alignment of proteins and the corresponding mRNA sequences into a codon alignment. Then the calculation of non-synonymous (Ka) and synonymous (Ks) substitution rates were carried out on the PAL2NAL website automatically by using the codeml program in PAML . By performing the EMBOSS Pairwise Alignment Algorithms on EBI http://www.ebi.ac.uk/Tools/emboss/align/, we analyzed the protein identities between paralogs of OsPTR.
We thank Professor Qifa Zhang for his encouragement and direction. We also are grateful to the reviewers and editors for their helpful comments and criticisms. This research was supported by grants from the National Special Key Project of China on Functional Genomics of Major Plants and Animals, and the National Natural Science Foundation of China.
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