BACKGROUND
venomous snakes possess one of the most sophisticated integrated weapons system in the natural world.
it has been hypothesized that the snake venom gland itself evolved in the mouth region as a consequence of an evolutionary change in the pancreatic trait, and consequently, some of the toxins should show strong affinities to pancreatic proteins  <cit> .
recent studies suggested that snake venom components such as phospholipase a <dig> have evolved in an accelerated manner to acquire their diverse physiological activities  <cit> .
most studies of snake venom glands have focused almost exclusively on the components of venom.
therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components?
to obtain a more comprehensive view of function of the venom gland, it is necessary for us to choose a proper model to study gene expression and toxin diversity in snake venom gland and perform comparative research with other species.
deinagkistrodon acutus  is a specific snake in southern china and many toxin components from the venom gland of d. acutus have been purified and characterized  <cit> .
more studies for d. acutus venom gland will promote the application of toxin in medicine.
to our best knowledge, there is little information about the d. acutus venom gland at the molecular level.
this problem is compounded by the limited number of annotated d. acutus nucleotide sequences currently deposited in the public databases .
for this reason, d. acutus should be a good model for use in studying gene expression and organogenesis in snake venom gland.
analysis of expressed sequence tags  is an efficient approach for gene discovery, expression profiling  <cit>  and development of resources useful for functional genomics studies.
for this purpose, we decided to adopt the strategy of large scale sequencing by constructing a cdna library from the venom gland of d. acutus.
thus we should be able to find relevant genes and investigate their function after screening and expressing the target genes.
knowledge of genes and proteins expressed in the various venom glands offers a potential resource to guide future investigations relevant to biology of venom gland and toxicology.
for instance, proteins secreted from the venom gland of d. acutus represent a subset of candidates potentially involved in local and systemic effects as pain, edema, bleeding and muscle necrosis.
moreover, gene cataloguing and profiling of the venom gland of d. acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for the elucidation mechanisms of action of toxins and the discovery of their antagonists  <cit> .
on the other hand, it will allow the identification of cellular functional transcripts that may represent a general panorama of the physiological events of the venom glands, surveying gene expression from the very specialized secretory tissue.
RESULTS
overview of ests from the venom gland of d. acutus
after discarding the poor-quality sequences,  <dig> high-quality ests were used to analyze gene expression profile in the venom gland of d. acutus.
the mean read length of ests was  <dig> bp .
subsequently, ests were clustered into  <dig> clusters, of which  <dig> clusters  associated with toxin function has been reported elsewhere  <cit> .
in this report, we discussed mainly  <dig> clusters  involved in the cellular functional transcripts and other novel sequences .
the distribution of all ests was followed:
twenty-five clusters consisted of more than  <dig> ests each, which represented the most abundant transcripts and encoded known proteins.
they constituted  <dig> % of the total clusters  including  <dig> % of total ests .
interestingly, half of the most abundant transcripts were previously reported metalloproteinase from venom gland of d. acutus  <cit> , indicating high prevalence.
of these  <dig> clusters, eight clusters are known genes that belong to housekeeping genes and two are toxin secretion related genes .
thirty-nine clusters consisted of 20– <dig> ests each and represented  <dig> % of the total clusters  and  <dig> % of the total ests .
of  <dig> clusters,  <dig> encoded non-toxin functional proteins, such as myosin, nadh dehydrogenase subunit, cytochrome oxidase subunit and calmodulin protein.
they are the second most abundant mrna transcripts in the venom gland of d. acutus.
seventy-five clusters contained 10– <dig> ests each, and represented  <dig> % of the total clusters  and  <dig> % of ests , of which  <dig> clusters encoded the genes for troponin, atpase subunits, retrotransposable-like elements and elongation factor 1-alpha, etc.
they are considered medium-sized clusters with relatively low prevalence.
the low abundant  <dig> clusters consisted of 2– <dig> ests each and constituted  <dig> % of the total clusters  and  <dig> % of the total ests .
they included many toxin coding genes, cellular functional transcripts and partial unknown protein e.g., jerdonitin, proline dehydrogenase and hypothetical proteins.
there are  <dig> unique ests representing  <dig> % of the total clusters  and  <dig> % of ests .
the occurrence rate of these clusters is only once in current sequenced ests.
they included cytokine-like molecule, zinc finger proteins, transport proteins and transcripts without hits to genbank non-redundant proteins  and nucleic acids databases .
the distribution of these cluster sizes are shown in figure  <dig>
the cdna library constructed is a non-normalized primary library without amplification, so the clone abundance or the cluster size presents the relative mrna population  <cit> .
about one-third of the total clones are singletons, and approximately one-fourth of the ests fit in clusters that are comprised of more than  <dig> ests, representing the complexity and specificity of the transcript population of the venom gland of d. acutus.
ests relevant to protein processing
a homologue of bothrops insularis calglandulin ef-hand protein family is identified at high abundance  in current library .
calglandulin ef-hand protein as a venom gland specific gene has been reported  <cit> .
it has several conserved ca2+ motifs and is expressed exclusively in snake venom glands from many species, but not secreted to the venom.
this protein family functions in the process of exporting toxins out of the cell and into the venom  <cit> , implying that it plays a fundamental role in toxin secretion process  <cit> .
in this library, three ef-hand protein families were found, of which two showed high identity with bothrops insularis ef-hand protein family and another showed homology only with mus musculus calmodulin.
the diversity of ef-hand proteins in the venom gland of d. acutus may suggest the complexity of toxin secretion activity.
the other high expression gene encodes the protein disulfide isomerase , which was represented by  <dig> ests in the library.
the pdi from d. acutus showed  <dig> % of identity with gallus gallus pdi.
the pdi is a redox protein responsible for disulfide bond assembly in the endoplasmatic reticulum.
we also found a significant frequency difference of cysteine residues between toxin protein and cellular functional proteins  in the venom gland of d. acutus, suggesting that the pdi plays a key role in toxin protein folding.
furthermore, heat shock proteins  are also identified  in this library including hsp <dig>  hsp <dig> and hsp <dig>  hsps are chaperon for protein refolding and degeneration, which is possibly important to toxin proteins regeneration.
there are many ribosomal proteins found in this library, which contributes to the high level of protein synthesis events.
a large number of ribosomal proteins therefore are needed for the toxins synthesis  <cit> .
several other identified transcripts can also shed light on the physiological aspects of the venom gland secretion style.
for instance, various clusters involved in transporter activity are found, e.g., ion transporters , nucleoside transporters  .
all these suggested that the venom gland of d. acutus is a highly specialized active organ that plays a central role in secreting toxins and polypeptides with powerful synthesis capabilities.
ests relevant to structural components and energy supply
there are abundant structural component transcripts expressed in the venom gland of d. acutus, encoding actin, troponin, calsequestrin and myosin .
interestingly, these cellular structural components from the venom gland are similar to ones from mammalian muscle tissue, which may indicate that the structure of the venom gland cavity is similar to muscle tissue and contributes to the venom gland contractile activity.
accordingly, it could be explained that creatine kinase expressed highly in the venom gland of d. acutus, accounting for  <dig> %  of all ests .
because creatine kinase is an important enzyme regulator of high-energy phosphate production and utilization within contractile tissues, high expression of the enzyme is adapted to energy need for gland contraction.
furthermore, abundant transcripts expressed in current library also involved in cytochrome b, cytochrome oxidase and nadh dehydrogenase, which are also needed to meet energy needs for toxin protein synthesis and gland contraction.
enzymes relevant to metabolism pathway
in this library, several enzymes in metabolic pathways such as glucose metabolism and nicotinate and nicotinamide metabolism were found .
in energy and material metabolism,  <dig> clusters sequences were identified to play a role in glucose metabolism.
we also identified that unigene uni <dig> and unigene uni <dig> code for the 5'-nucleotidase, which suggests that d. acutus may possess a functional pathway for purine metabolism and nicotinate and nicotinamide metabolism in the venom gland.
the 5'-nucleotidase participates not only in purine metabolism but also in nicotinate and nicotinamide metabolism.
snake envenomation employs three well-integrated strategies: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion.
purines  constitute the perfect multifunctional toxins, and evidently play a central role in all three envenomation strategies of most advanced snakes  <cit> .
ests relevant to other function
surprisingly,  <dig> clusters  encoding for reverse transcriptase were found in current library.
they are similar to reverse transcriptase from teleost line family sw <dig>  <cit> .
at the same time, we identified retrotransposable-like elements in this library , most of them similar to orf <dig> protein from a platemys spixii retrotransposon cr <dig>  <cit> .
so we could expect an intact retrotransposable structure in the d. acutus genome.
this specific retrotransposable element in the d. acutus genome may be adapted to environmental diversity and prey need.
so far, we still have not determined the complete functional categories of genes expressed in the venom gland of snake.
to give an overview of the major cellular roles, the number of partial mrna transcripts represented in each category is listed  based on molecular function of gene ontology  <cit> .
a major proportion  represent transcripts involved in the binding category, corresponding to  <dig> % of genes of molecular function and  <dig> % of total unigenes, respectively.
based on gene ontology function classification,  <dig> clusters  are assigned into the organizing principle of molecular function and  <dig> clusters  of biological process .
however, such an analysis only gave a hint of what the function might be and, in many cases, extensive biochemical and biological work is necessary to unambiguously identify a gene and its function  <cit> .
we have presented here an initial analysis of those relevant to physiological cellular proteins.
ests identifying no significant matches to known genes
there are  <dig> clusters  without significant homology to any known genes in genbank.
according to sequences discarding criteria , we could exclude the possibility that too short sequences lead to no hits.
the high abundant novel sequences represent a large number of unidentified genes, suggesting the complexity and diversity of genes expressed in the venom gland of d. acutus.
in addition, among those clusters without significant matches to known genes,  <dig> clusters have matches with the dbest database and  <dig> clusters with the hmmpfam database.
some of those clusters, such as unigene rfstca0_ <dig> y <dig> scf showed a putative toxin-related motif region of disintegrin, and unigene rfstda0_ <dig> y <dig> scf for conotoxin i-superfamily, indicated new toxins among those sequences.
the high abundance of these sequences might correspond to the unknown toxin genes stored in the venom gland of d. acutus.
further study of these novel genes expressed in this specialized organ could disclose the mechanism of toxin secretion and the evolution of the snake venom gland.
comparative analysis with other snakes venom glands
although several cdna libraries from the venomous gland of a few snakes have been reported and characterized  <cit> , analysis of transcripts from these cdna libraries seldom involve cellular functions as the main attention was focused on the toxin components.
many components of toxin in the venom gland have been identified  <cit> , but nerve growth factor  has not been identified in this library.
in contrast to previous reports  <cit> , we postulate an alternative possibility for not identifying ngf in this library: one is that the ngf might express under the specifically physiological conditions, such as in milking venom gland of d. acutus; another is that ngf is not a necessary component of toxin of d. acutus.
furthermore, a lot of clusters that may be involved in many physiological process of venom gland remained to be deciphered.
it is significant to study the gene expression of venom gland of snake on cellular structure and functional aspects, which will improve the study of some physiological process such as organogenesis, cell differentiation and protein synthesis.
alternatively, some secreted membrane proteins may represent antigens of potential importance to immune control.
phylogenetic analysis of toxin related genes in d. acutus
snake venom glands evolved once, at the base of the colubroid radiation, 60– <dig> million years ago, with extensive subsequent "evolutionary tinkering"  <cit> .
the advanced snakes  make up >80% of about  <dig> species of snake currently described, and contain all of the known venomous forms  <cit> .
generally in this library, toxin clusters match sequences from snake sources in database while the cellular functional transcripts are identified by its similarity to model organisms such as gallus gallus, homo sapiens and rattus norvegicus.
of note, although those transcripts of toxin are always phylogenetically closer from another snake, the average of similarities over toxins and cellular functional transcripts has no obvious difference.
from the view of evolution, we postulated that the toxins tend to diverge due to natural selective pressure to adapt to different environmental conditions .
whereas, most of the cellular components showing similarities with mammalian proteins, although they are usually phylogenetically distant, correspond to proteins of conserved functions among vertebrates and thus show higher homology  <cit> .
the origin and evolution of many toxin gene families in the advanced snakes have been researched extensively  <cit> .
among these toxin gene families, most were recruited into the advanced snakes before the split of elapids and viperids.
however, the phylogenetic analysis of a few other toxin gene families, such as phospholipase a <dig> and natriuretic toxins provide a clear evidence of an independent recruitment event.
because of limited toxin gene sequences in public databases, the origin and evolution of a number of toxin gene families remain unknown.
in this report, we analyzed the phylogeny of the group iii snake venom metalloproteinase and serine protease.
the group iii snake venom metalloproteinase consists of a proprotein, a metalloproteinase, a disintegrin and a cysteine-rich domain.
it inhibits the integrin receptor selectively.
figure  <dig> described the phylogenetic analysis of this group iii metalloproteinase in colubroid.
the phylogenetic model of this metalloproteinase is similar to crisp protein  <cit> , which was recruited into the advanced snakes before the split of elapids and viperids.
that is the advanced snake acquired the group iii metalloproteinase genes by an early single recruitment event.
subsequently, this metalloproteinase family evolved independently in elapids and viperids.
we also analyzed the phylogeny of the serine proteases in suborder colubroidea and similar results were shown .
but serine protease genes have not been identified in elapids, which suggested the gene loss events in the evolutionary process or insufficient sequences information of elapids.
CONCLUSIONS
this study identified and characterized the cellular functional transcripts in the venom gland of d. acutus extensively.
our aim is to develop a catalog of genes transcribed in this snake venom gland.
we hoped to discover as many toxin and cellular coding genes as possible by constructing five fraction sub-libraries.
the venom gland of d. acutus expresses many protein-coding sequences that are too divergent from those at present in genbank to provide identification.
moreover, we found as many unidentified protein-coding sequences as identified ones.
the prevalence distribution also provides a reasonable estimation of the actual frequency of these sequences in the venom gland of d. acutus.
furthermore, we have described a number of recognized molecules previously not known to be expressed in the venom gland of d. acutus, i.e., dehydrogenase and calglandulin ef-hand protein families.
though it is of relatively small size, analysis of this set of ests has yielded several kinds of useful information pertaining to the unmilked venom gland of d. acutus.
it is, therefore, likely that the generation of more sequence data will result in the identification of novel d. acutus genes.
in addition, many ests do not have significant database matches and these open up new avenues of exploration.
this report successfully provides evidence about the function of snake venom gland and a source of reptilian sequences.
gene cataloguing and profiling of the venom gland of d. acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissues.
so this study provides a first global view of the genetic programs for the venom gland of d. acutus described so far and an insight into molecular mechanism of toxin secreting in the very specialized organ.
materials and methods
library construction
a non-normalized cdna library was generated from the venom gland of d. acutus.
the d. acutus was captured from wuyi mountain, fujian province, china.
after killing the snake by cutting the head, the venom glands were recovered.
all tissues were immediately frozen in liquid nitrogen.
the total rna was isolated with trizol reagent  and the mrna was purified with oligotex mrna kits .
the cdna synthesized with superscipt ii-rt  and dna polymerase i , was flanked by ecor i adaptor  and treated by xho i .
the double strand cdna was extracted by electrophoresis separation with five fractions  and then cloned into ecor i and xho i digested pbluescript ii vector.
the plasmid was transformed into e. coli  to amplify the cdna.
est sequencing, data processing and bioinformatics analysis
the cdna clones were picked randomly.
plasmids were isolated according to a standard alkaline lysis protocol, and sequenced with megabace  <dig> .
base-calling was performed with phred  <cit> , the cutoff phred score was  <dig>  <cit> .
original sequences were generated by removing vector and e. coli dna sequences using cross-match  <cit> .
high-quality ests were assembled into contigs using phrap software  <cit> .
default settings were used except  <dig> bp minimum overlap and 99% identity.
to assign annotation to the assembled ests , these sequences were searched against the nr  and nt  for homology comparison using blastx and blastn  <cit> .
then those clusters without significant matches to known genes were compared to the dbest database by blast and pfam database by hmmpfam  <cit> .
all assembled sequences having the same annotation were further clustered into a unique gene.
based on gene ontology classification, we constructed a gene expression profile of the venom gland of d. acutus.
in order to comprehend the role of those transcripts played in the biochemical process, we assigned enzyme functions and enzyme commission numbers to reconstruct anabolic and catabolic pathways through linkage to biochemical pathways in the kegg database  <cit> .
the phylogenetic analysis of toxin protein families was conducted by mega  <dig>   <cit> .
authors' contributions
zb, lq participated in the design of study, carried out the experiments and the comparative analysis, and drafted the manuscript.
yw participated in the design of study.
zx, ly participated in the design of study and the data analysis.
hy, qp, sx participated in the data analysis.
yj, hs, yg conceived the study and participated in coordinate and help to draft the manuscript.
all authors have read and approved the paper.

 venomous snakes possess one of the most sophisticated integrated weapons system in the natural world. it has been hypothesized that the snake venom gland itself evolved in the mouth region as a consequence of an evolutionary change in the pancreatic trait, and consequently, some of the toxins should show strong affinities to pancreatic proteins. recent studies suggested that snake venom components such as phospholipase a have evolved in an accelerated manner to acquire their diverse physiological activities. most studies of snake venom glands have focused almost exclusively on the components of venom. therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components? to obtain a more comprehensive view of function of the venom gland, it is necessary for us to choose a proper model to study gene expression and toxin diversity in snake venom gland and perform comparative research with other species. deinagkistrodon acutus is a specific snake in southern china and many toxin components from the venom gland of d. acutus have been purified and characterized. more studies for d. acutus venom gland will promote the application of toxin in medicine. to our best knowledge, there is little information about the d. acutus venom gland at the molecular level. this problem is compounded by the limited number of annotated d. acutus nucleotide sequences currently deposited in the public databases. for this reason, d. acutus should be a good model for use in studying gene expression and organogenesis in snake venom gland. analysis of expressed sequence tags is an efficient approach for gene discovery, expression profiling and development of resources useful for functional genomics studies. for this purpose, we decided to adopt the strategy of large scale sequencing by constructing a cdna library from the venom gland of d. acutus. thus we should be able to find relevant genes and investigate their function after screening and expressing the target genes. knowledge of genes and proteins expressed in the various venom glands offers a potential resource to guide future investigations relevant to biology of venom gland and toxicology. for instance, proteins secreted from the venom gland of d. acutus represent a subset of candidates potentially involved in local and systemic effects as pain, edema, bleeding and muscle necrosis. moreover, gene cataloguing and profiling of the venom gland of d. acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for the elucidation mechanisms of action of toxins and the discovery of their antagonists. on the other hand, it will allow the identification of cellular functional transcripts that may represent a general panorama of the physiological events of the venom glands, surveying gene expression from the very specialized secretory tissue.  overview of ests from the venom gland of d. acutus after discarding the poor-quality sequences, high-quality ests were used to analyze gene expression profile in the venom gland of d. acutus. the mean read length of ests was bp. subsequently, ests were clustered into clusters, of which clusters associated with toxin function has been reported elsewhere. in this report, we discussed mainly clusters involved in the cellular functional transcripts and other novel sequences. the distribution of all ests was followed: twenty-five clusters consisted of more than ests each, which represented the most abundant transcripts and encoded known proteins. they constituted % of the total clusters including % of total ests. interestingly, half of the most abundant transcripts were previously reported metalloproteinase from venom gland of d. acutus, indicating high prevalence. of these clusters, eight clusters are known genes that belong to housekeeping genes and two are toxin secretion related genes. thirty-nine clusters consisted of 20– ests each and represented % of the total clusters and % of the total ests. of clusters, encoded non-toxin functional proteins, such as myosin, nadh dehydrogenase subunit, cytochrome oxidase subunit and calmodulin protein. they are the second most abundant mrna transcripts in the venom gland of d. acutus. seventy-five clusters contained 10– ests each, and represented % of the total clusters and % of ests, of which clusters encoded the genes for troponin, atpase subunits, retrotransposable-like elements and elongation factor 1-alpha, etc. they are considered medium-sized clusters with relatively low prevalence. the low abundant clusters consisted of 2– ests each and constituted % of the total clusters and % of the total ests. they included many toxin coding genes, cellular functional transcripts and partial unknown protein e.g., jerdonitin, proline dehydrogenase and hypothetical proteins. there are unique ests representing % of the total clusters and % of ests. the occurrence rate of these clusters is only once in current sequenced ests. they included cytokine-like molecule, zinc finger proteins, transport proteins and transcripts without hits to genbank non-redundant proteins and nucleic acids databases. the distribution of these cluster sizes are shown in figure the cdna library constructed is a non-normalized primary library without amplification, so the clone abundance or the cluster size presents the relative mrna population. about one-third of the total clones are singletons, and approximately one-fourth of the ests fit in clusters that are comprised of more than ests, representing the complexity and specificity of the transcript population of the venom gland of d. acutus. ests relevant to protein processing a homologue of bothrops insularis calglandulin ef-hand protein family is identified at high abundance in current library. calglandulin ef-hand protein as a venom gland specific gene has been reported. it has several conserved ca2+ motifs and is expressed exclusively in snake venom glands from many species, but not secreted to the venom. this protein family functions in the process of exporting toxins out of the cell and into the venom, implying that it plays a fundamental role in toxin secretion process. in this library, three ef-hand protein families were found, of which two showed high identity with bothrops insularis ef-hand protein family and another showed homology only with mus musculus calmodulin. the diversity of ef-hand proteins in the venom gland of d. acutus may suggest the complexity of toxin secretion activity. the other high expression gene encodes the protein disulfide isomerase, which was represented by ests in the library. the pdi from d. acutus showed % of identity with gallus gallus pdi. the pdi is a redox protein responsible for disulfide bond assembly in the endoplasmatic reticulum. we also found a significant frequency difference of cysteine residues between toxin protein and cellular functional proteins in the venom gland of d. acutus, suggesting that the pdi plays a key role in toxin protein folding. furthermore, heat shock proteins are also identified in this library including hsp hsp and hsp hsps are chaperon for protein refolding and degeneration, which is possibly important to toxin proteins regeneration. there are many ribosomal proteins found in this library, which contributes to the high level of protein synthesis events. a large number of ribosomal proteins therefore are needed for the toxins synthesis. several other identified transcripts can also shed light on the physiological aspects of the venom gland secretion style. for instance, various clusters involved in transporter activity are found, e.g., ion transporters, nucleoside transporters. all these suggested that the venom gland of d. acutus is a highly specialized active organ that plays a central role in secreting toxins and polypeptides with powerful synthesis capabilities. ests relevant to structural components and energy supply there are abundant structural component transcripts expressed in the venom gland of d. acutus, encoding actin, troponin, calsequestrin and myosin. interestingly, these cellular structural components from the venom gland are similar to ones from mammalian muscle tissue, which may indicate that the structure of the venom gland cavity is similar to muscle tissue and contributes to the venom gland contractile activity. accordingly, it could be explained that creatine kinase expressed highly in the venom gland of d. acutus, accounting for % of all ests. because creatine kinase is an important enzyme regulator of high-energy phosphate production and utilization within contractile tissues, high expression of the enzyme is adapted to energy need for gland contraction. furthermore, abundant transcripts expressed in current library also involved in cytochrome b, cytochrome oxidase and nadh dehydrogenase, which are also needed to meet energy needs for toxin protein synthesis and gland contraction. enzymes relevant to metabolism pathway in this library, several enzymes in metabolic pathways such as glucose metabolism and nicotinate and nicotinamide metabolism were found. in energy and material metabolism, clusters sequences were identified to play a role in glucose metabolism. we also identified that unigene uni and unigene uni code for the 5'-nucleotidase, which suggests that d. acutus may possess a functional pathway for purine metabolism and nicotinate and nicotinamide metabolism in the venom gland. the 5'-nucleotidase participates not only in purine metabolism but also in nicotinate and nicotinamide metabolism. snake envenomation employs three well-integrated strategies: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion. purines constitute the perfect multifunctional toxins, and evidently play a central role in all three envenomation strategies of most advanced snakes. ests relevant to other function surprisingly, clusters encoding for reverse transcriptase were found in current library. they are similar to reverse transcriptase from teleost line family sw. at the same time, we identified retrotransposable-like elements in this library, most of them similar to orf protein from a platemys spixii retrotransposon cr. so we could expect an intact retrotransposable structure in the d. acutus genome. this specific retrotransposable element in the d. acutus genome may be adapted to environmental diversity and prey need. so far, we still have not determined the complete functional categories of genes expressed in the venom gland of snake. to give an overview of the major cellular roles, the number of partial mrna transcripts represented in each category is listed based on molecular function of gene ontology. a major proportion represent transcripts involved in the binding category, corresponding to % of genes of molecular function and % of total unigenes, respectively. based on gene ontology function classification, clusters are assigned into the organizing principle of molecular function and clusters of biological process. however, such an analysis only gave a hint of what the function might be and, in many cases, extensive biochemical and biological work is necessary to unambiguously identify a gene and its function. we have presented here an initial analysis of those relevant to physiological cellular proteins. ests identifying no significant matches to known genes there are clusters without significant homology to any known genes in genbank. according to sequences discarding criteria, we could exclude the possibility that too short sequences lead to no hits. the high abundant novel sequences represent a large number of unidentified genes, suggesting the complexity and diversity of genes expressed in the venom gland of d. acutus. in addition, among those clusters without significant matches to known genes, clusters have matches with the dbest database and clusters with the hmmpfam database. some of those clusters, such as unigene rfstca0_ y scf showed a putative toxin-related motif region of disintegrin, and unigene rfstda0_ y scf for conotoxin i-superfamily, indicated new toxins among those sequences. the high abundance of these sequences might correspond to the unknown toxin genes stored in the venom gland of d. acutus. further study of these novel genes expressed in this specialized organ could disclose the mechanism of toxin secretion and the evolution of the snake venom gland. comparative analysis with other snakes venom glands although several cdna libraries from the venomous gland of a few snakes have been reported and characterized, analysis of transcripts from these cdna libraries seldom involve cellular functions as the main attention was focused on the toxin components. many components of toxin in the venom gland have been identified, but nerve growth factor has not been identified in this library. in contrast to previous reports, we postulate an alternative possibility for not identifying ngf in this library: one is that the ngf might express under the specifically physiological conditions, such as in milking venom gland of d. acutus; another is that ngf is not a necessary component of toxin of d. acutus. furthermore, a lot of clusters that may be involved in many physiological process of venom gland remained to be deciphered. it is significant to study the gene expression of venom gland of snake on cellular structure and functional aspects, which will improve the study of some physiological process such as organogenesis, cell differentiation and protein synthesis. alternatively, some secreted membrane proteins may represent antigens of potential importance to immune control. phylogenetic analysis of toxin related genes in d. acutus snake venom glands evolved once, at the base of the colubroid radiation, 60– million years ago, with extensive subsequent "evolutionary tinkering". the advanced snakes make up >80% of about species of snake currently described, and contain all of the known venomous forms. generally in this library, toxin clusters match sequences from snake sources in database while the cellular functional transcripts are identified by its similarity to model organisms such as gallus gallus, homo sapiens and rattus norvegicus. of note, although those transcripts of toxin are always phylogenetically closer from another snake, the average of similarities over toxins and cellular functional transcripts has no obvious difference. from the view of evolution, we postulated that the toxins tend to diverge due to natural selective pressure to adapt to different environmental conditions. whereas, most of the cellular components showing similarities with mammalian proteins, although they are usually phylogenetically distant, correspond to proteins of conserved functions among vertebrates and thus show higher homology. the origin and evolution of many toxin gene families in the advanced snakes have been researched extensively. among these toxin gene families, most were recruited into the advanced snakes before the split of elapids and viperids. however, the phylogenetic analysis of a few other toxin gene families, such as phospholipase a and natriuretic toxins provide a clear evidence of an independent recruitment event. because of limited toxin gene sequences in public databases, the origin and evolution of a number of toxin gene families remain unknown. in this report, we analyzed the phylogeny of the group iii snake venom metalloproteinase and serine protease. the group iii snake venom metalloproteinase consists of a proprotein, a metalloproteinase, a disintegrin and a cysteine-rich domain. it inhibits the integrin receptor selectively. figure described the phylogenetic analysis of this group iii metalloproteinase in colubroid. the phylogenetic model of this metalloproteinase is similar to crisp protein, which was recruited into the advanced snakes before the split of elapids and viperids. that is the advanced snake acquired the group iii metalloproteinase genes by an early single recruitment event. subsequently, this metalloproteinase family evolved independently in elapids and viperids. we also analyzed the phylogeny of the serine proteases in suborder colubroidea and similar results were shown. but serine protease genes have not been identified in elapids, which suggested the gene loss events in the evolutionary process or insufficient sequences information of elapids.  this study identified and characterized the cellular functional transcripts in the venom gland of d. acutus extensively. our aim is to develop a catalog of genes transcribed in this snake venom gland. we hoped to discover as many toxin and cellular coding genes as possible by constructing five fraction sub-libraries. the venom gland of d. acutus expresses many protein-coding sequences that are too divergent from those at present in genbank to provide identification. moreover, we found as many unidentified protein-coding sequences as identified ones. the prevalence distribution also provides a reasonable estimation of the actual frequency of these sequences in the venom gland of d. acutus. furthermore, we have described a number of recognized molecules previously not known to be expressed in the venom gland of d. acutus, i.e., dehydrogenase and calglandulin ef-hand protein families. though it is of relatively small size, analysis of this set of ests has yielded several kinds of useful information pertaining to the unmilked venom gland of d. acutus. it is, therefore, likely that the generation of more sequence data will result in the identification of novel d. acutus genes. in addition, many ests do not have significant database matches and these open up new avenues of exploration. this report successfully provides evidence about the function of snake venom gland and a source of reptilian sequences. gene cataloguing and profiling of the venom gland of d. acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissues. so this study provides a first global view of the genetic programs for the venom gland of d. acutus described so far and an insight into molecular mechanism of toxin secreting in the very specialized organ. materials and methods library construction a non-normalized cdna library was generated from the venom gland of d. acutus. the d. acutus was captured from wuyi mountain, fujian province, china. after killing the snake by cutting the head, the venom glands were recovered. all tissues were immediately frozen in liquid nitrogen. the total rna was isolated with trizol reagent and the mrna was purified with oligotex mrna kits. the cdna synthesized with superscipt ii-rt and dna polymerase i, was flanked by ecor i adaptor and treated by xho i. the double strand cdna was extracted by electrophoresis separation with five fractions and then cloned into ecor i and xho i digested pbluescript ii vector. the plasmid was transformed into e. coli to amplify the cdna. est sequencing, data processing and bioinformatics analysis the cdna clones were picked randomly. plasmids were isolated according to a standard alkaline lysis protocol, and sequenced with megabace. base-calling was performed with phred, the cutoff phred score was. original sequences were generated by removing vector and e. coli dna sequences using cross-match. high-quality ests were assembled into contigs using phrap software. default settings were used except bp minimum overlap and 99% identity. to assign annotation to the assembled ests, these sequences were searched against the nr and nt for homology comparison using blastx and blastn. then those clusters without significant matches to known genes were compared to the dbest database by blast and pfam database by hmmpfam. all assembled sequences having the same annotation were further clustered into a unique gene. based on gene ontology classification, we constructed a gene expression profile of the venom gland of d. acutus. in order to comprehend the role of those transcripts played in the biochemical process, we assigned enzyme functions and enzyme commission numbers to reconstruct anabolic and catabolic pathways through linkage to biochemical pathways in the kegg database. the phylogenetic analysis of toxin protein families was conducted by mega. authors' contributions zb, lq participated in the design of study, carried out the experiments and the comparative analysis, and drafted the manuscript. yw participated in the design of study. zx, ly participated in the design of study and the data analysis. hy, qp, sx participated in the data analysis. yj, hs, yg conceived the study and participated in coordinate and help to draft the manuscript. all authors have read and approved the paper. 
the snake venom gland is a specialized organ, which synthesizes and secretes the complex and abundant toxin proteins. though gene expression in the snake venom gland has been extensively studied, the focus has been on the components of the venom. therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components? to examine extensively the transcripts expressed in the venom gland of deinagkistrodon acutus and unveil the potential of its products on cellular structure and functional aspects, we generated expressed sequence tags from a non-normalized cdna library. gene cataloguing and profiling of the venom gland of deinagkistrodon acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissue. so this study provides a first global view of the genetic programs for the venom gland of deinagkistrodon acutus described so far and an insight into molecular mechanism of toxin secreting. as far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. by analyzing cellular functional transcripts, we found high expression of some venom related genes and gland-specific genes, such as calglandulin ef-hand protein gene and protein disulfide isomerase gene. the phylogenetic analysis of two snake venom toxin families of group iii metalloproteinase and serine protease in suborder colubroidea showed an early single recruitment event in the viperids evolutionary process. all ests were clustered into clusters, of which % of total ests belong to recognized toxin-coding sequences; % are similar to cellular transcripts; and % have no significant similarity to any known sequences. the transcripts of creatine kinase and nadh dehydrogenase were also identified at high level.
 the snake venom gland is a specialized organ, which synthesizes and secretes the complex and abundant toxin proteins. though gene expression in the snake venom gland has been extensively studied, the focus has been on the components of the venom. as far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components?  to examine extensively the transcripts expressed in the venom gland of deinagkistrodon acutus and unveil the potential of its products on cellular structure and functional aspects, we generated expressed sequence tags from a non-normalized cdna library. all ests were clustered into clusters, of which % of total ests belong to recognized toxin-coding sequences; % are similar to cellular transcripts; and % have no significant similarity to any known sequences. by analyzing cellular functional transcripts, we found high expression of some venom related genes and gland-specific genes, such as calglandulin ef-hand protein gene and protein disulfide isomerase gene. the transcripts of creatine kinase and nadh dehydrogenase were also identified at high level. moreover, abundant cellular structural proteins similar to mammalian muscle tissues were also identified. the phylogenetic analysis of two snake venom toxin families of group iii metalloproteinase and serine protease in suborder colubroidea showed an early single recruitment event in the viperids evolutionary process.  gene cataloguing and profiling of the venom gland of deinagkistrodon acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissue. so this study provides a first global view of the genetic programs for the venom gland of deinagkistrodon acutus described so far and an insight into molecular mechanism of toxin secreting. all sequences data reported in this paper have been submitted into the public database. 