BACKGROUND
the sequencing and analysis of expressed sequence tags  has been a primary tool for the discovery of novel genes in plants, especially in non-model plants for which full genome sequences are not currently available  <cit> .
est sequencing represents a rapid and cost-effective method for analyzing the transcribed regions of genomes.
est analysis is also a powerful tool for the discovery of genes involved in plant secondary metabolism.
the  <dig> gs flx sequencing technology has made est-based resources more readily accessible for non-model organism transcriptomes  <cit> .
our experimental focus for this study was glycyrrhiza uralensis fisch.
ex dc., which is one of the most ancient medicinal herbs and has been used as a chinese herbal medicine to treat infectious diseases for over  <dig>  years  <cit> .
this herb has been extensively studied and is widely used as a flavoring agent, medicament and tobacco additive.
many of the biological activities of the bioactive constituents of g. uralensis have been investigated, including the protection against hepatotoxicity  <cit> , anti-ulcer effects  <cit> , anti-inflammatory  <cit>  and anti-tumor promoting activities  <cit> .
this herb also exhibits antiviral activity against various dna and rna viruses, including herpes simplex virus  <cit> , hiv  <cit>  and severe acute respiratory syndrome -associated coronavirus  <cit> .
these biological activities of g. uralensis have been primarily attributed to two of its components, flavonoids and saponins.
our research interests primarily concern glycyrrhizin, an oleanane-type triterpene saponin and a well-known natural sweetener that is fifty times sweeter than sugar  <cit> .
although the various chemical and pharmacological properties of glycyrrhizin in g. uralensis have been extensively studied, the biosynthetic pathway of this compound remains poorly understood.
two functional genes encoding squalene synthase  have been isolated from g. uralensis  <cit> .
two cytochrome p <dig> genes have also been isolated from g. uralensis based on the traditional est sequencing method  <cit> .
cyp88d <dig>  a cytochrome p <dig> monooxygenase, was characterized by in vitro enzymatic activity assays and was shown to catalyze the oxidation of β-amyrin at c- <dig> to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate in the glycyrrhizin biosynthetic pathway  <cit> .
another cytochrome p <dig> from g. uralensis, cyp93e <dig>  possesses β-amyrin 24-hydroxylase activity in in vitro enzymatic activity assays  <cit> .
a functional β-amyrin synthase gene  has been isolated from g. glabra  <cit> .
thus far, only one glycosyltransferase in the glycyrrhiza genus, the isoflavonoid glucosyltransferase in g. echinata, has been identified  <cit> .
however, no progress has been made in the identification of the genes involved in the glycosylation of glycyrrhetinic acid to produce glycyrrhizin.
transcriptome sequencing would provide a foundation for detailed studies of gene expression and genetic connectivity with respect to plant secondary metabolism.
in our study, we constructed a cdna library using the vegetative organs of five-year-old wild g. uralensis cultivated from the city of yanchi in the ningxia province of china, one of the most famous areas for the production of wild g. uralensis.
the library was sequenced using the  <dig> gs flx platform and titanium reagents.
there are currently  <dig>  g. uralensis ests in the genbank  dbest database, which were determined using conventional sequencing techniques  <cit> .
in our study, we increased this collection with an additional  <dig>  ests generated from  <dig> gs flx titanium sequencing.
bioinformatic analyses indicated that almost all of the genes involved in the biosynthesis of the glycyrrhizin skeleton were within the combined est database, except for mevalonate kinase  and dxp synthase .
additionally, a pool of candidate genes for cytochrome p450s and glycosyltransferases was established, containing  <dig> and  <dig> unigenes, respectively.
finally, using an organ-specific expression pattern analysis, a few unigenes were selected as the candidates most likely to be responsible for glycyrrhizin skeleton modifications.
the method described here is a cost-effective technology for the identification of novel genes in non-model organisms that serve as medicinal plants.
furthermore, the ests and unigenes described in our study constitute an important resource for future studies of the molecular genetics and functional genomics of g. uralensis.
RESULTS
<dig> sequencing and assembly
a g. uralensis cdna library was constructed from a pool of mrna isolated from the vegetative organs of g. uralensis and was sequenced using the  <dig> gs flx platform and titanium regents.
after the initial quality filtering, this one-eighth sequencing run produced  <dig>  high-quality reads  with an average length of  <dig> bp  and a total length of  <dig>  mb.
of these hq reads,  <dig>  exceeded our minimal quality standards  and were used in the assembly.
the large portion of reads used after trimming and filtering indicates that most of the sequences were used in the assembly and that the quality of the data was considerably high.
an additional  <dig>  g. uralensis ests were downloaded from genbank  and combined with our  <dig> derived ests.
a summary of these two est datasets is given in table  <dig>  figure  <dig> illustrates the sequence length distributions of the ests derived from  <dig> sequencing and genbank.
assembly of the trimmed, size-selected ests produced  <dig>  contigs with a mean length of  <dig> bp and a range of  <dig> -  <dig>  bp, as well as an additional  <dig>  singletons, for a total of  <dig>  unigenes  .
the length distribution of the contigs is shown in figure  <dig>  the assembly produced a substantial number of large contigs .
approximately 16% of the unigenes  were composed of the  <dig> ests and guests data .
the  <dig> sequencing identified  <dig>  novel unigenes, bringing the total number of different g. uralensis unigenes in the databases to  <dig> .
this number should cover the vast majority of genes from this species, including those expressed at low levels.
ahq indicates high-quality
aunigenes includes contigs and singletons
for homology searches against known genes, unigenes longer than  <dig> bp are widely accepted as valid sources, making them sufficient for the effective assignment of functional annotations  <cit> .
most of the unigene sequences were longer than  <dig> bp and were valid in this study .
functional annotation
our annotation method was based on sequence homology searches and the annotations that accompanied them.
its aim was to capture the most informative and complete annotation possible.
additional file  <dig> shows the hit numbers and percentages relative to those of the major public databases.
these annotation statistics show all the unigenes annotated by the blast  <cit>  search against the public protein and nucleotide databases  where the e-value threshold was set at 1e- <dig>  of the  <dig>  unigenes,  <dig>  unigenes had at least one hit within these databases.
the remaining unigenes  that were not annotated likely comprise g. uralensis-specific genes, as well as genes with homologs in other species whose corresponding biological functions have not yet been investigated.
gene ontology classification
as a result of the completed genomic sequencing of the plant arabidopsis thaliana, the currently available expressed sequences have been invaluable in defining the correct components of the gene structure in this species  <cit> .
to obtain an overview of the gene functions of the ests from g. uralensis, the annotated unigenes were categorized according to gene ontology  on the basis of the tair go slim provided by tair  <cit> .
the g. uralensis unigenes were compared with the arabidopsis proteome  <cit>  using blastx  <cit> .
we used the annotations of each unigene to assign it to the go categories for molecular function, biological process and cellular component .
the go analysis showed that the functions of the identified genes are involved various biological processes.
a large number of hydrolases, kinases and transferases were annotated, which suggests that our study may allow for the identification of novel genes involved in the secondary metabolite synthesis pathways.
putative genes related to the biosynthesis of glycyrrhizin
in this study, our primary goal was to identify genes involved in the glycyrrhizin biosynthetic pathway   <cit> .
the biosynthesis of glycyrrhizin involves the synthesis of dimethylallyl diphosphate  and isopentenyl diphosphate , the biochemically active isoprene units of all terpenoids  <cit> .
this step is followed by the synthesis of the triterpene skeleton, also known as β-amyrin  <cit> , and then by a series of oxidative reactions and glucuronylations, which produce glycyrrhizin.
the precise order of the intermediate products is still unknown  <cit> .
ablast against the swissprot and kegg databases; benzyme code
in the early stage of active isoprene unit formation, plants have the ability to produce dmapp and ipp using two pathways, the mevalonate pathway  and the methylerythritol phosphate pathway .
in plants, these two pathways appear to be separate; enzymes of the mva pathway are found in the cytosol, whereas enzymes of the mep pathway are localized in plastids.
triterpenoids are known to be formed by the mva pathway because they are cytosolic products.
however, there are examples where the two pathways can act cooperatively to create a molecule  <cit> .
no progress has been made toward determining the precise source of isoprene units in glycyrrhizin biosynthesis.
using a blast  <cit>  search against the swissprot  <cit>  and kegg  <cit>  databases, we found the genes encoding all of the enzymes from both of these two pathways in the est database, except for mevalonate kinase , which is located in the mva pathway, and dxp synthase , which is located in the mep pathway.
in this study, we found all of the putative genes encoding the enzymes involved in the triterpene skeleton β-amyrin synthesis step: farnesyl diphosphate synthase , squalene synthase , squalene monooxygenase and β-amyrin synthase .
the enzymes involved in the biosyntheses of the isoprene unit and the triterpene skeleton are listed in table  <dig>  a list of putative unigenes involved in the glycyrrhizin biosynthetic pathway is shown in additional file  <dig>
cytochrome p <dig> and glycosyltransferase
glycyrrhizin is derived from the triterpene β-amyrin, which is an initial from product of thethe cyclization of  <dig>  3-oxidosqualene.
the subsequent steps in glycyrrhizin biosynthesis include a series of oxidative and glycosyl transfer reactions.
we have little knowledge of the later steps in the glycyrrhizin biosynthetic pathway, which include multiple oxidation and glycosylation steps that are catalyzed by enzymes from the cytochrome p <dig>  and glycosyltransferase superfamilies, respectively.
cytochrome p <dig> is a very large and diverse superfamily of hemoproteins that are found in all higher organisms  <cit> .
plant p450s catalyze many different reactions involved in the biosynthesis of secondary metabolites, including terpenoids  <cit> .
some members of the cyp <dig> and cyp <dig> families have been shown to act on β-amyrin or related triterpene substrates with unique reaction specificities  <cit> .
thus far, all known cytochrome p450s that act on triterpenes and sterols have been classified into two clans: the cyp <dig> clan and the cyp <dig> clan, which includes cyp <dig> and cyp <dig>  respectively  <cit> .
only two cyp genes of g. uralensis have been identified  <cit> .
an organ-specific transcript profiling approach was used in other studies to identify cyp88d <dig>  which catalyzes the oxidation of β-amyrin at c- <dig> to produce 11-oxo-β-amyrin in the glycyrrhizin biosynthetic pathway.
the expression profile of cyp88d <dig> was consistent with the organ-specific accumulation pattern of glycyrrhizin  <cit> ; a higher level of expression was seen in the root than in the stem and leaf.
by mining the est database, we found  <dig> unigenes  annotated as putative cyp genes, which were further classified into  <dig> cyp families and  <dig> subfamilies .
to narrow down the candidate cytochrome p450s, these unigenes were further screened according to their classification.
in the candidate p <dig> dataset, two unigenes  were annotated as cyp <dig>  while six unigenes  were annotated as cyp <dig>  in total,  <dig> contigs annotated as cytochrome p <dig> and belonging to the cyp <dig> and cyp <dig> clans , were chosen for organ-specific expression pattern assays .
contig <dig>  was exactly the same as the cyp88d <dig> gene according to the blast annotation results.
an additional  <dig> unigenes had a similar organ-specific expression pattern as cyp88d <dig>  including contig <dig> , contig <dig>  and contig <dig> .
however, additional experiments are needed to determine which of these unigenes participate in glycyrrhizin biosynthesis.
glycosyltransferases, a ubiquitous family of enzymes, catalyze reactions involving the transfer of a nucleotide-activated sugar moiety onto another molecule  <cit> .
these enzymes are encoded by a large multigene family; approximately  <dig> secondary metabolism glycosyltransferase genes have been identified in arabidopsis.
the conjugation of a sugar moiety to a substrate is called glycosylation, which is a process that contributes to the synthesis of glucidic polymers, glycoproteins and glycolipids.
glycosyltransferases often use specific substrates in the glycosylation reaction and are relevant for the synthesis of secondary metabolites.
no genes encoding relevant glycosyltransferases have been identified in glycyrrhiza.
using blast searches, approximately  <dig> unigenes  in our study showed sequence similarities to glycosyltransferase  in the kegg database.
according to the go category analysis, these unigenes were classified into  <dig> categories .
among these categories,  <dig> unigenes  encoded for udp-glucosyltransferases, which is obviously involved in the biosynthesis of secondary metabolites.
we also pick up unigenes annotated as glucuronosyltransferases because glycyrrhizin is composed of aglycone glycyrrhetic acid and two glucuronic acid units.
we found  <dig> unigenes  that encoded glucuronosyltransferases, and it is possible that these are involved in the last steps of glycyrrhizin biosynthesis.
from these two categories,  <dig> contigs were chosen  for organ-specific expression pattern analysis by real-time pcr .
the expression patterns of  <dig> glycosyltransferase unigenes were similar to that of cyp88d <dig>  these glycosyltransferases included contig <dig> , contig <dig> , contig <dig> , contig <dig> , contig <dig>  and contig <dig> .
these contigs were regarded as candidate glycosyltransferases that encode the enzymes responsible for glycyrrhizin biosynthesis and will be the subject of further study.
we did not select singletons that were annotated as cytochrome p450s or glycosyltransferases for the organ-specific expression pattern analysis because of the high content of glycyrrhizin in the glycyrrhiza plant   <cit> .
on the other hand,  <dig> ests were annotated as cyp88d <dig> , which is a known cytochrome p <dig> gene in the glycyrrhizin biosynthetic pathway.
the lists of candidate unigenes for cytochrome p450s and glycosyltransferases are found in additional files  <dig> and  <dig>  respectively.
CONCLUSIONS
our study established a high-quality est database for g. uralensis using  <dig> gs flx titanium sequencing technology.
with this work, we initiated a large-scale investigation of the transcriptome of g. uralensis in terms of functional genomics, molecular biology and biochemistry.
a large number of novel candidate genes involved in glycyrrhizin biosynthesis, including cytochrome p450s and glycosyltransferases, were identified in our est dataset.
the information from these ests represents a significant contribution toward the exploration of the molecular mechanisms of glycyrrhizin biosynthesis.
more importantly, a few candidate genes encoding the enzymes responsible for glycyrrhizin skeleton modifications were obtained by screening functional annotations and by organ-specific expression pattern analyses.

 the sequencing and analysis of expressed sequence tags has been a primary tool for the discovery of novel genes in plants, especially in non-model plants for which full genome sequences are not currently available. est sequencing represents a rapid and cost-effective method for analyzing the transcribed regions of genomes. est analysis is also a powerful tool for the discovery of genes involved in plant secondary metabolism. the gs flx sequencing technology has made est-based resources more readily accessible for non-model organism transcriptomes. our experimental focus for this study was glycyrrhiza uralensis fisch. ex dc., which is one of the most ancient medicinal herbs and has been used as a chinese herbal medicine to treat infectious diseases for over years. this herb has been extensively studied and is widely used as a flavoring agent, medicament and tobacco additive. many of the biological activities of the bioactive constituents of g. uralensis have been investigated, including the protection against hepatotoxicity, anti-ulcer effects, anti-inflammatory and anti-tumor promoting activities. this herb also exhibits antiviral activity against various dna and rna viruses, including herpes simplex virus, hiv and severe acute respiratory syndrome -associated coronavirus. these biological activities of g. uralensis have been primarily attributed to two of its components, flavonoids and saponins. our research interests primarily concern glycyrrhizin, an oleanane-type triterpene saponin and a well-known natural sweetener that is fifty times sweeter than sugar. although the various chemical and pharmacological properties of glycyrrhizin in g. uralensis have been extensively studied, the biosynthetic pathway of this compound remains poorly understood. two functional genes encoding squalene synthase have been isolated from g. uralensis. two cytochrome p genes have also been isolated from g. uralensis based on the traditional est sequencing method. cyp88d a cytochrome p monooxygenase, was characterized by in vitro enzymatic activity assays and was shown to catalyze the oxidation of β-amyrin at c- to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate in the glycyrrhizin biosynthetic pathway. another cytochrome p from g. uralensis, cyp93e possesses β-amyrin 24-hydroxylase activity in in vitro enzymatic activity assays. a functional β-amyrin synthase gene has been isolated from g. glabra. thus far, only one glycosyltransferase in the glycyrrhiza genus, the isoflavonoid glucosyltransferase in g. echinata, has been identified. however, no progress has been made in the identification of the genes involved in the glycosylation of glycyrrhetinic acid to produce glycyrrhizin. transcriptome sequencing would provide a foundation for detailed studies of gene expression and genetic connectivity with respect to plant secondary metabolism. in our study, we constructed a cdna library using the vegetative organs of five-year-old wild g. uralensis cultivated from the city of yanchi in the ningxia province of china, one of the most famous areas for the production of wild g. uralensis. the library was sequenced using the gs flx platform and titanium reagents. there are currently g. uralensis ests in the genbank dbest database, which were determined using conventional sequencing techniques. in our study, we increased this collection with an additional ests generated from gs flx titanium sequencing. bioinformatic analyses indicated that almost all of the genes involved in the biosynthesis of the glycyrrhizin skeleton were within the combined est database, except for mevalonate kinase and dxp synthase. additionally, a pool of candidate genes for cytochrome p450s and glycosyltransferases was established, containing and unigenes, respectively. finally, using an organ-specific expression pattern analysis, a few unigenes were selected as the candidates most likely to be responsible for glycyrrhizin skeleton modifications. the method described here is a cost-effective technology for the identification of novel genes in non-model organisms that serve as medicinal plants. furthermore, the ests and unigenes described in our study constitute an important resource for future studies of the molecular genetics and functional genomics of g. uralensis.  sequencing and assembly a g. uralensis cdna library was constructed from a pool of mrna isolated from the vegetative organs of g. uralensis and was sequenced using the gs flx platform and titanium regents. after the initial quality filtering, this one-eighth sequencing run produced high-quality reads with an average length of bp and a total length of mb. of these hq reads, exceeded our minimal quality standards and were used in the assembly. the large portion of reads used after trimming and filtering indicates that most of the sequences were used in the assembly and that the quality of the data was considerably high. an additional g. uralensis ests were downloaded from genbank and combined with our derived ests. a summary of these two est datasets is given in table figure illustrates the sequence length distributions of the ests derived from sequencing and genbank. assembly of the trimmed, size-selected ests produced contigs with a mean length of bp and a range of - bp, as well as an additional singletons, for a total of unigenes. the length distribution of the contigs is shown in figure the assembly produced a substantial number of large contigs. approximately 16% of the unigenes were composed of the ests and guests data. the sequencing identified novel unigenes, bringing the total number of different g. uralensis unigenes in the databases to. this number should cover the vast majority of genes from this species, including those expressed at low levels. ahq indicates high-quality aunigenes includes contigs and singletons for homology searches against known genes, unigenes longer than bp are widely accepted as valid sources, making them sufficient for the effective assignment of functional annotations. most of the unigene sequences were longer than bp and were valid in this study. functional annotation our annotation method was based on sequence homology searches and the annotations that accompanied them. its aim was to capture the most informative and complete annotation possible. additional file shows the hit numbers and percentages relative to those of the major public databases. these annotation statistics show all the unigenes annotated by the blast search against the public protein and nucleotide databases where the e-value threshold was set at 1e- of the unigenes, unigenes had at least one hit within these databases. the remaining unigenes that were not annotated likely comprise g. uralensis-specific genes, as well as genes with homologs in other species whose corresponding biological functions have not yet been investigated. gene ontology classification as a result of the completed genomic sequencing of the plant arabidopsis thaliana, the currently available expressed sequences have been invaluable in defining the correct components of the gene structure in this species. to obtain an overview of the gene functions of the ests from g. uralensis, the annotated unigenes were categorized according to gene ontology on the basis of the tair go slim provided by tair. the g. uralensis unigenes were compared with the arabidopsis proteome using blastx. we used the annotations of each unigene to assign it to the go categories for molecular function, biological process and cellular component. the go analysis showed that the functions of the identified genes are involved various biological processes. a large number of hydrolases, kinases and transferases were annotated, which suggests that our study may allow for the identification of novel genes involved in the secondary metabolite synthesis pathways. putative genes related to the biosynthesis of glycyrrhizin in this study, our primary goal was to identify genes involved in the glycyrrhizin biosynthetic pathway. the biosynthesis of glycyrrhizin involves the synthesis of dimethylallyl diphosphate and isopentenyl diphosphate, the biochemically active isoprene units of all terpenoids. this step is followed by the synthesis of the triterpene skeleton, also known as β-amyrin, and then by a series of oxidative reactions and glucuronylations, which produce glycyrrhizin. the precise order of the intermediate products is still unknown. ablast against the swissprot and kegg databases; benzyme code in the early stage of active isoprene unit formation, plants have the ability to produce dmapp and ipp using two pathways, the mevalonate pathway and the methylerythritol phosphate pathway. in plants, these two pathways appear to be separate; enzymes of the mva pathway are found in the cytosol, whereas enzymes of the mep pathway are localized in plastids. triterpenoids are known to be formed by the mva pathway because they are cytosolic products. however, there are examples where the two pathways can act cooperatively to create a molecule. no progress has been made toward determining the precise source of isoprene units in glycyrrhizin biosynthesis. using a blast search against the swissprot and kegg databases, we found the genes encoding all of the enzymes from both of these two pathways in the est database, except for mevalonate kinase, which is located in the mva pathway, and dxp synthase, which is located in the mep pathway. in this study, we found all of the putative genes encoding the enzymes involved in the triterpene skeleton β-amyrin synthesis step: farnesyl diphosphate synthase, squalene synthase, squalene monooxygenase and β-amyrin synthase. the enzymes involved in the biosyntheses of the isoprene unit and the triterpene skeleton are listed in table a list of putative unigenes involved in the glycyrrhizin biosynthetic pathway is shown in additional file cytochrome p and glycosyltransferase glycyrrhizin is derived from the triterpene β-amyrin, which is an initial from product of thethe cyclization of 3-oxidosqualene. the subsequent steps in glycyrrhizin biosynthesis include a series of oxidative and glycosyl transfer reactions. we have little knowledge of the later steps in the glycyrrhizin biosynthetic pathway, which include multiple oxidation and glycosylation steps that are catalyzed by enzymes from the cytochrome p and glycosyltransferase superfamilies, respectively. cytochrome p is a very large and diverse superfamily of hemoproteins that are found in all higher organisms. plant p450s catalyze many different reactions involved in the biosynthesis of secondary metabolites, including terpenoids. some members of the cyp and cyp families have been shown to act on β-amyrin or related triterpene substrates with unique reaction specificities. thus far, all known cytochrome p450s that act on triterpenes and sterols have been classified into two clans: the cyp clan and the cyp clan, which includes cyp and cyp respectively. only two cyp genes of g. uralensis have been identified. an organ-specific transcript profiling approach was used in other studies to identify cyp88d which catalyzes the oxidation of β-amyrin at c- to produce 11-oxo-β-amyrin in the glycyrrhizin biosynthetic pathway. the expression profile of cyp88d was consistent with the organ-specific accumulation pattern of glycyrrhizin; a higher level of expression was seen in the root than in the stem and leaf. by mining the est database, we found unigenes annotated as putative cyp genes, which were further classified into cyp families and subfamilies. to narrow down the candidate cytochrome p450s, these unigenes were further screened according to their classification. in the candidate p dataset, two unigenes were annotated as cyp while six unigenes were annotated as cyp in total, contigs annotated as cytochrome p and belonging to the cyp and cyp clans, were chosen for organ-specific expression pattern assays. contig was exactly the same as the cyp88d gene according to the blast annotation results. an additional unigenes had a similar organ-specific expression pattern as cyp88d including contig, contig and contig. however, additional experiments are needed to determine which of these unigenes participate in glycyrrhizin biosynthesis. glycosyltransferases, a ubiquitous family of enzymes, catalyze reactions involving the transfer of a nucleotide-activated sugar moiety onto another molecule. these enzymes are encoded by a large multigene family; approximately secondary metabolism glycosyltransferase genes have been identified in arabidopsis. the conjugation of a sugar moiety to a substrate is called glycosylation, which is a process that contributes to the synthesis of glucidic polymers, glycoproteins and glycolipids. glycosyltransferases often use specific substrates in the glycosylation reaction and are relevant for the synthesis of secondary metabolites. no genes encoding relevant glycosyltransferases have been identified in glycyrrhiza. using blast searches, approximately unigenes in our study showed sequence similarities to glycosyltransferase in the kegg database. according to the go category analysis, these unigenes were classified into categories. among these categories, unigenes encoded for udp-glucosyltransferases, which is obviously involved in the biosynthesis of secondary metabolites. we also pick up unigenes annotated as glucuronosyltransferases because glycyrrhizin is composed of aglycone glycyrrhetic acid and two glucuronic acid units. we found unigenes that encoded glucuronosyltransferases, and it is possible that these are involved in the last steps of glycyrrhizin biosynthesis. from these two categories, contigs were chosen for organ-specific expression pattern analysis by real-time pcr. the expression patterns of glycosyltransferase unigenes were similar to that of cyp88d these glycosyltransferases included contig, contig, contig, contig, contig and contig. these contigs were regarded as candidate glycosyltransferases that encode the enzymes responsible for glycyrrhizin biosynthesis and will be the subject of further study. we did not select singletons that were annotated as cytochrome p450s or glycosyltransferases for the organ-specific expression pattern analysis because of the high content of glycyrrhizin in the glycyrrhiza plant. on the other hand, ests were annotated as cyp88d, which is a known cytochrome p gene in the glycyrrhizin biosynthetic pathway. the lists of candidate unigenes for cytochrome p450s and glycosyltransferases are found in additional files and respectively.  our study established a high-quality est database for g. uralensis using gs flx titanium sequencing technology. with this work, we initiated a large-scale investigation of the transcriptome of g. uralensis in terms of functional genomics, molecular biology and biochemistry. a large number of novel candidate genes involved in glycyrrhizin biosynthesis, including cytochrome p450s and glycosyltransferases, were identified in our est dataset. the information from these ests represents a significant contribution toward the exploration of the molecular mechanisms of glycyrrhizin biosynthesis. more importantly, a few candidate genes encoding the enzymes responsible for glycyrrhizin skeleton modifications were obtained by screening functional annotations and by organ-specific expression pattern analyses. 
due to limited genomic and transcriptomic data, the biosynthetic pathway of glycyrrhizin, the major bioactive compound in g. uralensis, is currently unclear. identification of candidate genes involved in the glycyrrhizin biosynthetic pathway will significantly contribute to the understanding of the biosynthetic and medicinal chemistry of this compound. using the gs flx platform and titanium reagents, our study provides a high-quality est database for g. uralensis. the cytochrome p candidate genes were classified into cyp families, while the glycosyltransferase candidate genes were classified into categories by go analysis. based on the est analysis, novel candidate genes related to the secondary metabolite pathway of glycyrrhizin, including novel genes encoding cytochrome p450s and glycosyltransferases, were found. we used the gs flx platform and titanium regents to produce a substantial expressed sequence tag dataset from the vegetative organs of g. uralensis. to identify novel genes that encode cytochrome p enzymes and glycosyltransferases, which are related to glycyrrhizin metabolism, a total of and unigenes were found to be homologous to cytochrome p450s and glycosyltransferases, respectively. with the assistance of organ-specific expression pattern analysis, unigenes encoding cytochrome p450s and unigenes encoding glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis. a total of unique gene elements representing approximately independent transcripts were annotated using blast searches against the swissprot, kegg, tair, nr and nt databases. finally, cytochrome p enzymes and glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis through an organ-specific expression pattern analysis based on real-time pcr. ests were combined with the g. uralensis ests in genbank.
 glycyrrhiza uralensis is one of the most popular medicinal plants in the world and is also widely used in the flavoring of food and tobacco. due to limited genomic and transcriptomic data, the biosynthetic pathway of glycyrrhizin, the major bioactive compound in g. uralensis, is currently unclear. identification of candidate genes involved in the glycyrrhizin biosynthetic pathway will significantly contribute to the understanding of the biosynthetic and medicinal chemistry of this compound.  we used the gs flx platform and titanium regents to produce a substantial expressed sequence tag dataset from the vegetative organs of g. uralensis. a total of ests with an average read length of bp were generated. ests were combined with the g. uralensis ests in genbank. the combined ests were assembled into unique sequences. a total of unique gene elements representing approximately independent transcripts were annotated using blast searches against the swissprot, kegg, tair, nr and nt databases. the assembled sequences were annotated with gene names and gene ontology terms. with respect to the genes related to glycyrrhizin metabolism, genes encoding enzymes of the total steps of the glycyrrhizin skeleton synthesis pathway were found. to identify novel genes that encode cytochrome p enzymes and glycosyltransferases, which are related to glycyrrhizin metabolism, a total of and unigenes were found to be homologous to cytochrome p450s and glycosyltransferases, respectively. the cytochrome p candidate genes were classified into cyp families, while the glycosyltransferase candidate genes were classified into categories by go analysis. finally, cytochrome p enzymes and glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis through an organ-specific expression pattern analysis based on real-time pcr.  using the gs flx platform and titanium reagents, our study provides a high-quality est database for g. uralensis. based on the est analysis, novel candidate genes related to the secondary metabolite pathway of glycyrrhizin, including novel genes encoding cytochrome p450s and glycosyltransferases, were found. with the assistance of organ-specific expression pattern analysis, unigenes encoding cytochrome p450s and unigenes encoding glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis. 