BACKGROUND
peanut  is one of most important and widespread oil crops.
one of the major problems in peanut production worldwide is aflatoxin contamination, which is of great concern in peanut as this toxin can cause teratogenic and carcinogenic effects in animal and human.
infection of peanut by aspergillus flavus occurs not only in post-harvest but also in pre-harvest conditions  <cit> .
several biotic  and abiotic  factors are known to affect pre-harvest aflatoxin contamination, while the late season drought  which predispose peanut to aflatoxin contamination  <cit>  is more important in the semi-arid tropics  <cit> .
irrigation in late season can reduce peanut pre-harvest aflatoxin contamination, but this cultural practice seems to be impractical in some areas, especially in semi-arid and arid areas.
enhancing host plant resistance to pre-harvest a. flavus invasion and aflatoxin contamination is considered to be the most cost-effective control measure.
in the past decades, peanut cultivars with natural pre-harvest resistance to aflatoxin production have been identified through field screening  <cit> .
however, the agronomic traits of these varieties have been very poor for the direct commercial utility.
the progress in transferring the resistance genes from these resistant lines into commercial cultivars has been slow, due to lack of understanding of the resistance mechanism and markers associated with resistance  <cit> .
although drought stress is known to predispose peanut to aflatoxin contamination  <cit> , limited researches were reported on the mechanism of late season drought stress aggravating the a. flavus infection.
dorner et al   <cit>  observed that drought stress could decrease the capacity of peanut seeds to produce phytoalexins, and thus resulted in higher aflatoxin contamination.
the active water of seeds is the most important factor controlling the capacity of seeds to produce phytoalexins  <cit> .
luo et al   <cit>  used a microarray of  <dig> unigenes to investigate the up/down regulated gene profiles in peanut cultivar a <dig>  which is drought tolerant and resistant to pre-harvest aflatoxin contamination, and identified  <dig> unigenes that were potentially associated with drought tolerance or that responded to a. parasiticus challenge.
nevertheless, the significance of these unigenes in pre-harvest infection of peanut pods by aspergillus is incomplete without knowledge of their functions.
studies to understand host resistance mechanisms in maize and peanut against a. flavus infection and aflatoxin contamination indicate that proteins are a major factor contributing to kernel resistance  <cit> .
proteins serve as the bridge between genetic information encoded in the genome and the phenotype.
proteomics analysis reveals the plasticity of gene expression as it allows global analysis of gene products and physiological states of plant under particular conditions.
the objectives of this research were to:  compare the differential expression of proteins of resistant and susceptible peanut cultivars in response to a. flavus challenge under drought stress;  identify seed proteins associated with resistance to pre-harvest aflatoxin contamination in peanut.
in this study, a total of  <dig> differentially expressed proteins were identified and  <dig> proteins associated with pre-harvested aflatoxin contamination were further characterized by maldi-tof ms and their expression profiles were validated by real-time rt-pcr.
the identification of these potential proteins associated with the aflatoxin resistance in peanut could be useful in programmes on developing peanut varieties with resistant to pre-harvest aflatoxin contamination.
RESULTS
aflatoxin accumulation analysis in seeds of resistant and susceptible cultivars
seed aflatoxin b <dig> levels from the resistant cultivar  and susceptible cultivars  had baseline levels  under well-watered conditions, and no difference between the two cultivars was found .
under drought stress conditions, the seed aflatoxin b <dig> level in both yj- <dig> and yueyou  <dig> increased.
the level of aflatoxin b <dig> increased to  <dig> ppb and  <dig> ppb in yj- <dig> and yueyou  <dig> respectively under drought stress.
after artificial inoculation treatment with a. flavus under drought stress, the aflatoxin b <dig> level in seeds of the infected cultivar yj- <dig> increased to  <dig> ppb, whereas the level in the infected cultivar yueyou  <dig> increased to  <dig> ppb, suggesting that aflatoxin b <dig> accumulation in the susceptible cultivar yueyou  <dig> was around 14-fold compared to the resistant cultivar yj- <dig>  yj- <dig> exhibited a significant level of resistance to pre-harvest aflatoxin contamination.
these results are in agreement with several earlier reports of resistance in peanut  <cit> .
comparison of seed proteomic profiles between resistant and susceptible cultivars under a. flavus challenge and drought stress
to investigate the seed protein profiles, we carried out 2-de analysis of the proteins from six sample groups as described in the methods section.
due to the lower resolution at the anodal and cathodal ends of the first dimension tube gels, only the gel region where the pi ranged from  <dig> to  <dig> was further analyzed.
for each treatment, 2-de gels were run in three replicates.
more than  <dig> protein spots were repeatedly detected on coomassie brilliant blue g- <dig> -stained gels using image master  <dig>  software across all the samples  and the reproducibility of all gels were over  <dig> % .
a comparison of 2-de images revealed that there were both qualitative and quantitative differences in resistant or susceptible cultivars under the three treatment conditions .
under the well-watered condition, the 2-de gel of resistant cultivar yj- <dig> showed  <dig> high quality spots , while  <dig> unique,  <dig> up-regulated,  <dig> down-regulated and  <dig> disappeared spots were induced by drought stress,  <dig> unique,  <dig> up-regulated,  <dig> down- regulated and  <dig> disappeared spots were induced by a. flavus infection under drought stress .
the 2-de protein profiles of the susceptible cultivar  showed a similar differential expression pattern responsive to drought stress and a. flavus infection, but the number of differentially expressed spots was less than that of the resistant cultivar .
five unique,  <dig> up-regulated,  <dig> down-regulated and  <dig> disappeared spots were induced by drought stress, while  <dig> unique,  <dig> up-regulated,  <dig> down-regulated and  <dig> disappeared spots were induced by a. flavus infection under drought stress in susceptible cultivar yueyou  <dig> .
to investigate the host proteins responsive to a. flavus infection, a comparison was conducted with 2-de images of total seed proteins from the resistant cultivar  and the susceptible cultivar  with a. flavus infection under drought stress .
about  <dig> spots that showed differential expression in all analytical gels under a. flavus attack were identified.
among those,  <dig> protein spots that consistently exhibited unique, increased or decreased in abundance and at least four fold differences in spot intensity in gel of resistant cultivar  with a. flavus infection under drought stress, compared with gel of the susceptible cultivar  received the same treatment.
of these, five protein spots  with unique expression, six protein spots  with an up-regulated trend, and one protein spots  with a down-regulated trend in the resistant cultivar  by a. flavus infection under drought stress were selected for ms analysis.
the enlargements of the  <dig> differentially expressed proteins were shown in figure  <dig>
identification of the differentially expressed proteins related to resistance to pre-harvest aflatoxin contamination
all of the twelve differentially expressed proteins were excised and analyzed by maldi-tof-ms to identify their putative functions.
after searching against the green plant protein database in ncbi, all these protein spots were successfully identified by pmf analysis and matched known plant proteins.
those proteins and their annotated functions are listed in table  <dig>  since there are relatively few known peanut proteins and genomic sequences available, only three proteins matched peanut proteins.
among the twelve selected proteins, four were related to stress response: low molecular weight heat shock protein precursor , oxalate oxidase , trypsin inhibitor  and l-ascorbate peroxidase  <dig>  os07g <dig> , cdkd <dig>  and rio kinase  were signaling components.
sap domain-containing protein ,  <dig> s ribosomal protein l <dig>  and putative  <dig> s ribosomal protein s <dig>  were related to regulation of transcription.
pii protein  and iso-ara h <dig>  were storage protein.
a: spot number; b: theoretical molecular weight/isoelectric point; c: number of matched peptides; d: sequence coverage.
gene transcription profile analysis by real time rt-pcr
to validate the expression of the twelve identified proteins at transcription level, total rnas from six samples  were extracted and analyzed by real time rt-pcr.
the primer pairs used for real time rt-pcr were designed based on nucleotide sequences in ncbi databases and shown in table  <dig> the actin gene was chosen as internal control.
figure  <dig> shows the expression patterns of the twelve genes in the resistant cultivar  and the susceptible cultivar  under well-watered , drought stress and a. flavus infection accompanied with drought stress on the 50th days after treatments.
the results demonstrated that, of the five genes identified as the unique expressed group , s <dig> and s <dig> showed higher expression levels in the cv.
yj- <dig> than in the cv.
yueyou <dig>  s <dig> showed similar and the remaining two showed lower.
of the six proteins identified as the up-regulated group , four genes  showed higher expression levels in the resistant cultivar with a. flavus infection under drought stress.
in contrast, two genes  showed no correlation between mrna and protein expression levels.
one gene  identified in the down-regulated group, showed the identical level of transcript abundance in both resistant and susceptible cultivars with a. flavus infection plus drought stress.
discussion
in this study, proteins showing differentially expressed profiles in the resistant and susceptible cultivars with a. flavus infection under drought stress were identified by using a proteomic approach.
around  <dig> protein spots identified for quantitative analyses of differentially regulated proteins responsive to a. falvus attack, and the number of protein spots was more than that in earlier reports by liang et al   <cit>  and kottapalli et al   <cit> .
we have identified  <dig> protein spots which significantly increased or decreased in response to a. flavus infection under drought stress in resistant cultivar  versus susceptible cultivar.
these proteins could be divided into four functional groups including defense response, signaling components, regulation of transcription and storage protein.
os07g <dig>  with transferase and kinase activity is a key protein in biosynthetic process  <cit> .
cdkd <dig>  is involved in the phosphorylation of proteins and regulation of cell cycle  <cit> .
oxalate oxidase  belongs to the germin-like family of proteins and catalyzes the degradation of oxalic acid to produce carbon dioxide and hydrogen peroxide  <cit> .
reports of oxalate oxidase activity in response to pathogen attack have received considerable attention as it possibly plays a role in plant defense  <cit> .
in plants, pii protein  is a nuclear-encoded plastid protein  <cit>  and can be involved in the regulation of nitrogen metabolism  <cit> .
sap domain-containing protein  was a dna binding protein and its physiological roles remain to be unknown.
in this study, these five proteins had unique expression in resistant cultivars and completely absent in the susceptible cultivar in response to a. flavus infection under drought stress, or under only drought stress condition.
these proteins were, therefore, considered to be encoded by candidate resistance-related genes potentially involved in resistance to preharvest aflatoxin contamination.
heat shock proteins ,  <dig> s ribosomal protein ,  <dig> s ribosomal protein  and iso-ara h <dig>  were up-regulated in both cultivars only in a. flavus infection under drought stress condition, but the expression level in the resistant cultivar was higher than in susceptible cultivar.
heat shock proteins  are the most well-known stress related proteins in plants which are induced in response to a number of different stresses.
hsp can play a role as chaperons which are involved in correct folding of proteins and protect them from denaturing under stress condition  <cit> .
in this study, hsp proteins could only be observed in peanut seeds upon a. flavus attack under drought conditions.
this result was contradictive with those of chen et al   <cit> , in which they reported that hsp proteins were constitutively expressed and up-regulated in resistant maize lines versus susceptible lines  <cit> .
both  <dig> s ribosomal protein  and putative  <dig> s ribosomal protein  are structural constituents of ribosome with rna binding function, and play essential roles in translation processes  <cit> .
the transcripts of ribosomal proteins in leaves of arabidopsis plants were up-regulated under both drought and heat stress conditions  <cit> .
the significant up-regulation of two ribosomal proteins suggested that one of the major effects of pre-harvest a. flavus infection in peanut is imposed on protein synthesis.
iso-ara h <dig> , a peanut seed storage protein, shows significant homology to known peanut allergen, arah <dig>  <cit> .
the significant increase of iso-ara h <dig> in resistant cultivar compared with susceptible cultivar under a. flavus infection showed that iso-ara  <dig>  might be related to pre-harvest aflatoxin contamination.
l-ascorbate peroxidase  is a stress-responsive protein  <cit> , and is involved in the metabolism of h2o <dig> in higher plants  <cit> .
previous reports on peanut  <cit>  and maize  <cit>  showed l-ascorbate peroxidase were up-regulated by both a. parasticus and drought stress.
rio kinase  has kinase catalytic activity and is involved in atp binding  <cit> .
in this study, l-ascorbate peroxidase  and rio kinase  were detected only in the resistant cultivar under well-watered conditions, and were up-regulated under drought stress conditions and a. flavus attack under drought stress conditions.
in the susceptible cultivar, however, the two proteins were up-regulated only under a. flavus attack accompanied with drought stress.
this result was consistent with previous studies  <cit> .
this indicated that the two proteins  might contribute to increasing the resistance to pre-harvest aflatoxin contamination in the resistant cultivar.
trypsin inhibitor , a constitutively expressed antifungal protein, was observed at high expression levels in resistant peanut cultivars  <cit>  and maize lines  <cit> , but was at low or undetectable levels in susceptible cultivars and lines.
however, in this study, there was no differential expression in both cultivars under well-watered and drought stress conditions, but down-regulation of trypsin inhibitor was observed when challenged by a. flavus under drought stress in resistant cultivar.
the true reason of down-regulation of trypsin inhibitor in our experiment remains unknown.
the functional distribution of unique and up-regulated proteins in resistant cultivar  also showed that most of the proteins affected were defense-related proteins, protein synthesis, and regulation of transcription.
a. flavus infection in pre-harvested peanut seeds resulted in expression of six new proteins, no information of which was available in database.
three of them  were detectable only in resistant cultivar, and three proteins  were markedly up-regulated in resistant cultivar.
in addition, in this study, seven selected proteins for mrna expression study showed up-regulation in both mrna and protein expression, although it has been reported that the correlation between transcription and translation is known to be less than 50%  <cit> .
CONCLUSIONS
in conclusion, pre-harvest aflatoxin-resistance trait was characterized as a quantitative trait.
development of peanut cultivars with resistance to pre-harvest aflatoxin contamination would be a long-term selection program.
this study reports the first proteome analysis to identify resistance-associated protein such as low molecular weight heat shock protein, oso7g <dig>  pii protein, cdk <dig>  oxalate oxidase, sap domain-containing protein, rio kinase, l-ascorbate peroxidase, iso-ara h <dig>   <dig> s ribosomal protein,  <dig> s ribosomal, which may be associated with resistance to pre-harvest aflatoxin contamination in peanut.
more detailed analysis of the identified proteins is in progress to further characterize their possible functional roles in resistance to pre-harvested aflatoxin contamination.

 peanut is one of most important and widespread oil crops. one of the major problems in peanut production worldwide is aflatoxin contamination, which is of great concern in peanut as this toxin can cause teratogenic and carcinogenic effects in animal and human. infection of peanut by aspergillus flavus occurs not only in post-harvest but also in pre-harvest conditions. several biotic and abiotic factors are known to affect pre-harvest aflatoxin contamination, while the late season drought which predispose peanut to aflatoxin contamination is more important in the semi-arid tropics. irrigation in late season can reduce peanut pre-harvest aflatoxin contamination, but this cultural practice seems to be impractical in some areas, especially in semi-arid and arid areas. enhancing host plant resistance to pre-harvest a. flavus invasion and aflatoxin contamination is considered to be the most cost-effective control measure. in the past decades, peanut cultivars with natural pre-harvest resistance to aflatoxin production have been identified through field screening. however, the agronomic traits of these varieties have been very poor for the direct commercial utility. the progress in transferring the resistance genes from these resistant lines into commercial cultivars has been slow, due to lack of understanding of the resistance mechanism and markers associated with resistance. although drought stress is known to predispose peanut to aflatoxin contamination, limited researches were reported on the mechanism of late season drought stress aggravating the a. flavus infection. dorner et al observed that drought stress could decrease the capacity of peanut seeds to produce phytoalexins, and thus resulted in higher aflatoxin contamination. the active water of seeds is the most important factor controlling the capacity of seeds to produce phytoalexins. luo et al used a microarray of unigenes to investigate the up/down regulated gene profiles in peanut cultivar a which is drought tolerant and resistant to pre-harvest aflatoxin contamination, and identified unigenes that were potentially associated with drought tolerance or that responded to a. parasiticus challenge. nevertheless, the significance of these unigenes in pre-harvest infection of peanut pods by aspergillus is incomplete without knowledge of their functions. studies to understand host resistance mechanisms in maize and peanut against a. flavus infection and aflatoxin contamination indicate that proteins are a major factor contributing to kernel resistance. proteins serve as the bridge between genetic information encoded in the genome and the phenotype. proteomics analysis reveals the plasticity of gene expression as it allows global analysis of gene products and physiological states of plant under particular conditions. the objectives of this research were to: compare the differential expression of proteins of resistant and susceptible peanut cultivars in response to a. flavus challenge under drought stress; identify seed proteins associated with resistance to pre-harvest aflatoxin contamination in peanut. in this study, a total of differentially expressed proteins were identified and proteins associated with pre-harvested aflatoxin contamination were further characterized by maldi-tof ms and their expression profiles were validated by real-time rt-pcr. the identification of these potential proteins associated with the aflatoxin resistance in peanut could be useful in programmes on developing peanut varieties with resistant to pre-harvest aflatoxin contamination.  aflatoxin accumulation analysis in seeds of resistant and susceptible cultivars seed aflatoxin b levels from the resistant cultivar and susceptible cultivars had baseline levels under well-watered conditions, and no difference between the two cultivars was found. under drought stress conditions, the seed aflatoxin b level in both yj- and yueyou increased. the level of aflatoxin b increased to ppb and ppb in yj- and yueyou respectively under drought stress. after artificial inoculation treatment with a. flavus under drought stress, the aflatoxin b level in seeds of the infected cultivar yj- increased to ppb, whereas the level in the infected cultivar yueyou increased to ppb, suggesting that aflatoxin b accumulation in the susceptible cultivar yueyou was around 14-fold compared to the resistant cultivar yj- yj- exhibited a significant level of resistance to pre-harvest aflatoxin contamination. these results are in agreement with several earlier reports of resistance in peanut. comparison of seed proteomic profiles between resistant and susceptible cultivars under a. flavus challenge and drought stress to investigate the seed protein profiles, we carried out 2-de analysis of the proteins from six sample groups as described in the methods section. due to the lower resolution at the anodal and cathodal ends of the first dimension tube gels, only the gel region where the pi ranged from to was further analyzed. for each treatment, 2-de gels were run in three replicates. more than protein spots were repeatedly detected on coomassie brilliant blue g- -stained gels using image master software across all the samples and the reproducibility of all gels were over %. a comparison of 2-de images revealed that there were both qualitative and quantitative differences in resistant or susceptible cultivars under the three treatment conditions. under the well-watered condition, the 2-de gel of resistant cultivar yj- showed high quality spots, while unique, up-regulated, down-regulated and disappeared spots were induced by drought stress, unique, up-regulated, down- regulated and disappeared spots were induced by a. flavus infection under drought stress. the 2-de protein profiles of the susceptible cultivar showed a similar differential expression pattern responsive to drought stress and a. flavus infection, but the number of differentially expressed spots was less than that of the resistant cultivar. five unique, up-regulated, down-regulated and disappeared spots were induced by drought stress, while unique, up-regulated, down-regulated and disappeared spots were induced by a. flavus infection under drought stress in susceptible cultivar yueyou. to investigate the host proteins responsive to a. flavus infection, a comparison was conducted with 2-de images of total seed proteins from the resistant cultivar and the susceptible cultivar with a. flavus infection under drought stress. about spots that showed differential expression in all analytical gels under a. flavus attack were identified. among those, protein spots that consistently exhibited unique, increased or decreased in abundance and at least four fold differences in spot intensity in gel of resistant cultivar with a. flavus infection under drought stress, compared with gel of the susceptible cultivar received the same treatment. of these, five protein spots with unique expression, six protein spots with an up-regulated trend, and one protein spots with a down-regulated trend in the resistant cultivar by a. flavus infection under drought stress were selected for ms analysis. the enlargements of the differentially expressed proteins were shown in figure identification of the differentially expressed proteins related to resistance to pre-harvest aflatoxin contamination all of the twelve differentially expressed proteins were excised and analyzed by maldi-tof-ms to identify their putative functions. after searching against the green plant protein database in ncbi, all these protein spots were successfully identified by pmf analysis and matched known plant proteins. those proteins and their annotated functions are listed in table since there are relatively few known peanut proteins and genomic sequences available, only three proteins matched peanut proteins. among the twelve selected proteins, four were related to stress response: low molecular weight heat shock protein precursor, oxalate oxidase, trypsin inhibitor and l-ascorbate peroxidase os07g, cdkd and rio kinase were signaling components. sap domain-containing protein, s ribosomal protein l and putative s ribosomal protein s were related to regulation of transcription. pii protein and iso-ara h were storage protein. a: spot number; b: theoretical molecular weight/isoelectric point; c: number of matched peptides; d: sequence coverage. gene transcription profile analysis by real time rt-pcr to validate the expression of the twelve identified proteins at transcription level, total rnas from six samples were extracted and analyzed by real time rt-pcr. the primer pairs used for real time rt-pcr were designed based on nucleotide sequences in ncbi databases and shown in table the actin gene was chosen as internal control. figure shows the expression patterns of the twelve genes in the resistant cultivar and the susceptible cultivar under well-watered, drought stress and a. flavus infection accompanied with drought stress on the 50th days after treatments. the results demonstrated that, of the five genes identified as the unique expressed group, s and s showed higher expression levels in the cv. yj- than in the cv. yueyou s showed similar and the remaining two showed lower. of the six proteins identified as the up-regulated group, four genes showed higher expression levels in the resistant cultivar with a. flavus infection under drought stress. in contrast, two genes showed no correlation between mrna and protein expression levels. one gene identified in the down-regulated group, showed the identical level of transcript abundance in both resistant and susceptible cultivars with a. flavus infection plus drought stress. discussion in this study, proteins showing differentially expressed profiles in the resistant and susceptible cultivars with a. flavus infection under drought stress were identified by using a proteomic approach. around protein spots identified for quantitative analyses of differentially regulated proteins responsive to a. falvus attack, and the number of protein spots was more than that in earlier reports by liang et al and kottapalli et al. we have identified protein spots which significantly increased or decreased in response to a. flavus infection under drought stress in resistant cultivar versus susceptible cultivar. these proteins could be divided into four functional groups including defense response, signaling components, regulation of transcription and storage protein. os07g with transferase and kinase activity is a key protein in biosynthetic process. cdkd is involved in the phosphorylation of proteins and regulation of cell cycle. oxalate oxidase belongs to the germin-like family of proteins and catalyzes the degradation of oxalic acid to produce carbon dioxide and hydrogen peroxide. reports of oxalate oxidase activity in response to pathogen attack have received considerable attention as it possibly plays a role in plant defense. in plants, pii protein is a nuclear-encoded plastid protein and can be involved in the regulation of nitrogen metabolism. sap domain-containing protein was a dna binding protein and its physiological roles remain to be unknown. in this study, these five proteins had unique expression in resistant cultivars and completely absent in the susceptible cultivar in response to a. flavus infection under drought stress, or under only drought stress condition. these proteins were, therefore, considered to be encoded by candidate resistance-related genes potentially involved in resistance to preharvest aflatoxin contamination. heat shock proteins, s ribosomal protein, s ribosomal protein and iso-ara h were up-regulated in both cultivars only in a. flavus infection under drought stress condition, but the expression level in the resistant cultivar was higher than in susceptible cultivar. heat shock proteins are the most well-known stress related proteins in plants which are induced in response to a number of different stresses. hsp can play a role as chaperons which are involved in correct folding of proteins and protect them from denaturing under stress condition. in this study, hsp proteins could only be observed in peanut seeds upon a. flavus attack under drought conditions. this result was contradictive with those of chen et al, in which they reported that hsp proteins were constitutively expressed and up-regulated in resistant maize lines versus susceptible lines. both s ribosomal protein and putative s ribosomal protein are structural constituents of ribosome with rna binding function, and play essential roles in translation processes. the transcripts of ribosomal proteins in leaves of arabidopsis plants were up-regulated under both drought and heat stress conditions. the significant up-regulation of two ribosomal proteins suggested that one of the major effects of pre-harvest a. flavus infection in peanut is imposed on protein synthesis. iso-ara h, a peanut seed storage protein, shows significant homology to known peanut allergen, arah. the significant increase of iso-ara h in resistant cultivar compared with susceptible cultivar under a. flavus infection showed that iso-ara might be related to pre-harvest aflatoxin contamination. l-ascorbate peroxidase is a stress-responsive protein, and is involved in the metabolism of h2o in higher plants. previous reports on peanut and maize showed l-ascorbate peroxidase were up-regulated by both a. parasticus and drought stress. rio kinase has kinase catalytic activity and is involved in atp binding. in this study, l-ascorbate peroxidase and rio kinase were detected only in the resistant cultivar under well-watered conditions, and were up-regulated under drought stress conditions and a. flavus attack under drought stress conditions. in the susceptible cultivar, however, the two proteins were up-regulated only under a. flavus attack accompanied with drought stress. this result was consistent with previous studies. this indicated that the two proteins might contribute to increasing the resistance to pre-harvest aflatoxin contamination in the resistant cultivar. trypsin inhibitor, a constitutively expressed antifungal protein, was observed at high expression levels in resistant peanut cultivars and maize lines, but was at low or undetectable levels in susceptible cultivars and lines. however, in this study, there was no differential expression in both cultivars under well-watered and drought stress conditions, but down-regulation of trypsin inhibitor was observed when challenged by a. flavus under drought stress in resistant cultivar. the true reason of down-regulation of trypsin inhibitor in our experiment remains unknown. the functional distribution of unique and up-regulated proteins in resistant cultivar also showed that most of the proteins affected were defense-related proteins, protein synthesis, and regulation of transcription. a. flavus infection in pre-harvested peanut seeds resulted in expression of six new proteins, no information of which was available in database. three of them were detectable only in resistant cultivar, and three proteins were markedly up-regulated in resistant cultivar. in addition, in this study, seven selected proteins for mrna expression study showed up-regulation in both mrna and protein expression, although it has been reported that the correlation between transcription and translation is known to be less than 50%.  in conclusion, pre-harvest aflatoxin-resistance trait was characterized as a quantitative trait. development of peanut cultivars with resistance to pre-harvest aflatoxin contamination would be a long-term selection program. this study reports the first proteome analysis to identify resistance-associated protein such as low molecular weight heat shock protein, oso7g pii protein, cdk oxalate oxidase, sap domain-containing protein, rio kinase, l-ascorbate peroxidase, iso-ara h s ribosomal protein, s ribosomal, which may be associated with resistance to pre-harvest aflatoxin contamination in peanut. more detailed analysis of the identified proteins is in progress to further characterize their possible functional roles in resistance to pre-harvested aflatoxin contamination. 
the objective of this study was to identify resistance-associated proteins in response to a. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. to identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar and a susceptible cultivar under well-watered condition, drought stress, and a. flavus infection with drought stress. a total of spots showed differential expression between resistant and susceptible cultivars in response to a. flavus attack under drought stress. six protein spots including low molecular weight heat shock protein precursor, rio kinase, l-ascorbate peroxidase, iso-ara h s ribosomal protein l and putative s ribosomal s were significantly up-regulated in the resistant cultivar challenged by a. flavus under drought stress. a significant decrease or down regulation of trypsin inhibitor caused by a. flavus in the resistant cultivar was also observed. in summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination. some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. pre-harvest infection of peanuts by aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. however, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism. among these spots, protein spots that consistently exhibited an altered expression were screened by image master software and successfully identified by maldi-tof ms. five protein spots, including oso7g pii protein, cdk oxalate oxidase, sap domain-containing protein, were uniquely expressed in the resistant cultivar. in addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time rt-pcr analysis.
 pre-harvest infection of peanuts by aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. however, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism.  the objective of this study was to identify resistance-associated proteins in response to a. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. to identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar and a susceptible cultivar under well-watered condition, drought stress, and a. flavus infection with drought stress. a total of spots showed differential expression between resistant and susceptible cultivars in response to a. flavus attack under drought stress. among these spots, protein spots that consistently exhibited an altered expression were screened by image master software and successfully identified by maldi-tof ms. five protein spots, including oso7g pii protein, cdk oxalate oxidase, sap domain-containing protein, were uniquely expressed in the resistant cultivar. six protein spots including low molecular weight heat shock protein precursor, rio kinase, l-ascorbate peroxidase, iso-ara h s ribosomal protein l and putative s ribosomal s were significantly up-regulated in the resistant cultivar challenged by a. flavus under drought stress. a significant decrease or down regulation of trypsin inhibitor caused by a. flavus in the resistant cultivar was also observed. in addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time rt-pcr analysis.  in summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination. 