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Desiccation survival in an Antarctic nematode: molecular analysis using expressed sequenced tags.

Adhikari BN, Wall DH, Adams BJ - BMC Genomics (2009)

Bottom Line: The results indicate that the transcriptome contains a group of transcripts from diverse functional areas.Expression profiling of 14 selected genes by quantitative Real-time PCR showed 9 genes significantly up-regulated, 3 down-regulated and 2 continuously expressed in response to desiccation.The type of transcript analysis performed in this study sets the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in nematodes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA. adhikaribn@hotmail.com

ABSTRACT

Background: Nematodes are the dominant soil animals in Antarctic Dry Valleys and are capable of surviving desiccation and freezing in an anhydrobiotic state. Genes induced by desiccation stress have been successfully enumerated in nematodes; however we have little knowledge of gene regulation by Antarctic nematodes which can survive multiple environmental stresses. To address this problem we investigated the genetic responses of a nematode species, Plectus murrayi, that is capable of tolerating Antarctic environmental extremes, in particular desiccation and freezing. In this study, we provide the first insight into the desiccation induced transcriptome of an Antarctic nematode through cDNA library construction and suppressive subtractive hybridization.

Results: We obtained 2,486 expressed sequence tags (ESTs) from 2,586 clones derived from the cDNA library of desiccated P. murrayi. The 2,486 ESTs formed 1,387 putative unique transcripts of which 523 (38%) had matches in the model-nematode Caenorhabditis elegans, 107 (7%) in nematodes other than C. elegans, 153 (11%) in non-nematode organisms and 605 (44%) had no significant match to any sequences in the current databases. The 1,387 unique transcripts were functionally classified by using Gene Ontology (GO) hierarchy and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results indicate that the transcriptome contains a group of transcripts from diverse functional areas. The subtractive library of desiccated nematodes showed 80 transcripts differentially expressed during desiccation stress, of which 28% were metabolism related, 19% were involved in environmental information processing, 28% involved in genetic information processing and 21% were novel transcripts. Expression profiling of 14 selected genes by quantitative Real-time PCR showed 9 genes significantly up-regulated, 3 down-regulated and 2 continuously expressed in response to desiccation.

Conclusion: The establishment of a desiccation EST collection for Plectus murrayi, a useful model in assessing the structural, physiological, biochemical and genetic aspects of multiple stress tolerance, is an important step in understanding the genome level response of this nematode to desiccation stress. The type of transcript analysis performed in this study sets the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in nematodes.

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Quantitative Real-time PCR analysis of gene expression in Plectus murrayi in response to desiccation. Values were determined using qRT-PCR and represents relative expression of genes from desiccated to undesiccated nematodes (control). The relative expression of the target gene (Pm-alh: aldehyde dehydrogenase; Pm-tps: trehalose-6-phosphate synthase; Pm-gpx: glutathione peroxidise; Pm-afp: antifreeze protein; Pm-hsp-70: heat shock protein 70; Pm-lea: late embryogenesis abundant protein; Pm-gk: glycerol kinase; Pm-ms: malate synthase; Pm-gsy: glycogen synthase; Pm-hsp-90: heat shock protein 90; Pm-rpl-4: ribosomal protein-4; Pm-desc-1: novel protein I; Pm-desc-2: novel protein II; Pm-gst-1: glutathione s-transferase 1,) normalized to Pm-18s:18S rRNA and relative to the expression of control, was calculated for each sample using the 2-ΔΔCT method [87]. Gene expression was determined in each sample using three independent technical replicates. A transcript with relative abundance of one is equivalent to the abundance of 18S rRNA. Bars represent standard errors calculated from three replicates of each experiment. *Significant difference (P < 0.05) from control.
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Figure 3: Quantitative Real-time PCR analysis of gene expression in Plectus murrayi in response to desiccation. Values were determined using qRT-PCR and represents relative expression of genes from desiccated to undesiccated nematodes (control). The relative expression of the target gene (Pm-alh: aldehyde dehydrogenase; Pm-tps: trehalose-6-phosphate synthase; Pm-gpx: glutathione peroxidise; Pm-afp: antifreeze protein; Pm-hsp-70: heat shock protein 70; Pm-lea: late embryogenesis abundant protein; Pm-gk: glycerol kinase; Pm-ms: malate synthase; Pm-gsy: glycogen synthase; Pm-hsp-90: heat shock protein 90; Pm-rpl-4: ribosomal protein-4; Pm-desc-1: novel protein I; Pm-desc-2: novel protein II; Pm-gst-1: glutathione s-transferase 1,) normalized to Pm-18s:18S rRNA and relative to the expression of control, was calculated for each sample using the 2-ΔΔCT method [87]. Gene expression was determined in each sample using three independent technical replicates. A transcript with relative abundance of one is equivalent to the abundance of 18S rRNA. Bars represent standard errors calculated from three replicates of each experiment. *Significant difference (P < 0.05) from control.

Mentions: In order to validate our differential gene expression results and obtain more refined gene expression data, we designed gene-specific primers for 14 transcripts selected from Table 2 and analyzed their expression using quantitative real-time PCR (qRT-PCR) (Fig. 3). These genes were chosen to represent a variety of functional classifications. Among these 14 transcripts, 9 were significantly induced (fold-change > 2.0×, P value < 0.05) and 3 genes were reduced (fold-change > 2.0×, P value < 0.05) in response to desiccation. Significant desiccation-induced gene expression change ranged from -6.50-fold for antifreeze protein to 26.77-fold for trehalose-6-phosphate synthase. Heat shock protein 70 and 90 were also weakly induced (fold-change 1.7 and 1.94×), but lacked significant statistical support P > 0.05). Among the DE transcripts, putative homologs to trehalose 6-phosphate synthase protein showed highest induction (fold-change 26.77×, P value < 0.05) followed by the putative homolog to glycerol kinase (fold-change 25.04×, P value < 0.05). Interestingly, there was significant reduction and induction on the level of expression of two novel transcripts, [GenBank: FK670306 and FK670310] (fold-change -10.3× and 16.71× respectively, P < 0.05), suggestive of their possible roles in desiccation tolerance (Fig. 3). The mRNA copy number was calculated using an absolute quantification method [44]. There was no significant difference (P value < 0.05) in copy number of transcripts encoding Hsp70 and Hsp90 on desiccated and control nematodes (Additional file 2).


Desiccation survival in an Antarctic nematode: molecular analysis using expressed sequenced tags.

Adhikari BN, Wall DH, Adams BJ - BMC Genomics (2009)

Quantitative Real-time PCR analysis of gene expression in Plectus murrayi in response to desiccation. Values were determined using qRT-PCR and represents relative expression of genes from desiccated to undesiccated nematodes (control). The relative expression of the target gene (Pm-alh: aldehyde dehydrogenase; Pm-tps: trehalose-6-phosphate synthase; Pm-gpx: glutathione peroxidise; Pm-afp: antifreeze protein; Pm-hsp-70: heat shock protein 70; Pm-lea: late embryogenesis abundant protein; Pm-gk: glycerol kinase; Pm-ms: malate synthase; Pm-gsy: glycogen synthase; Pm-hsp-90: heat shock protein 90; Pm-rpl-4: ribosomal protein-4; Pm-desc-1: novel protein I; Pm-desc-2: novel protein II; Pm-gst-1: glutathione s-transferase 1,) normalized to Pm-18s:18S rRNA and relative to the expression of control, was calculated for each sample using the 2-ΔΔCT method [87]. Gene expression was determined in each sample using three independent technical replicates. A transcript with relative abundance of one is equivalent to the abundance of 18S rRNA. Bars represent standard errors calculated from three replicates of each experiment. *Significant difference (P < 0.05) from control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667540&req=5

Figure 3: Quantitative Real-time PCR analysis of gene expression in Plectus murrayi in response to desiccation. Values were determined using qRT-PCR and represents relative expression of genes from desiccated to undesiccated nematodes (control). The relative expression of the target gene (Pm-alh: aldehyde dehydrogenase; Pm-tps: trehalose-6-phosphate synthase; Pm-gpx: glutathione peroxidise; Pm-afp: antifreeze protein; Pm-hsp-70: heat shock protein 70; Pm-lea: late embryogenesis abundant protein; Pm-gk: glycerol kinase; Pm-ms: malate synthase; Pm-gsy: glycogen synthase; Pm-hsp-90: heat shock protein 90; Pm-rpl-4: ribosomal protein-4; Pm-desc-1: novel protein I; Pm-desc-2: novel protein II; Pm-gst-1: glutathione s-transferase 1,) normalized to Pm-18s:18S rRNA and relative to the expression of control, was calculated for each sample using the 2-ΔΔCT method [87]. Gene expression was determined in each sample using three independent technical replicates. A transcript with relative abundance of one is equivalent to the abundance of 18S rRNA. Bars represent standard errors calculated from three replicates of each experiment. *Significant difference (P < 0.05) from control.
Mentions: In order to validate our differential gene expression results and obtain more refined gene expression data, we designed gene-specific primers for 14 transcripts selected from Table 2 and analyzed their expression using quantitative real-time PCR (qRT-PCR) (Fig. 3). These genes were chosen to represent a variety of functional classifications. Among these 14 transcripts, 9 were significantly induced (fold-change > 2.0×, P value < 0.05) and 3 genes were reduced (fold-change > 2.0×, P value < 0.05) in response to desiccation. Significant desiccation-induced gene expression change ranged from -6.50-fold for antifreeze protein to 26.77-fold for trehalose-6-phosphate synthase. Heat shock protein 70 and 90 were also weakly induced (fold-change 1.7 and 1.94×), but lacked significant statistical support P > 0.05). Among the DE transcripts, putative homologs to trehalose 6-phosphate synthase protein showed highest induction (fold-change 26.77×, P value < 0.05) followed by the putative homolog to glycerol kinase (fold-change 25.04×, P value < 0.05). Interestingly, there was significant reduction and induction on the level of expression of two novel transcripts, [GenBank: FK670306 and FK670310] (fold-change -10.3× and 16.71× respectively, P < 0.05), suggestive of their possible roles in desiccation tolerance (Fig. 3). The mRNA copy number was calculated using an absolute quantification method [44]. There was no significant difference (P value < 0.05) in copy number of transcripts encoding Hsp70 and Hsp90 on desiccated and control nematodes (Additional file 2).

Bottom Line: The results indicate that the transcriptome contains a group of transcripts from diverse functional areas.Expression profiling of 14 selected genes by quantitative Real-time PCR showed 9 genes significantly up-regulated, 3 down-regulated and 2 continuously expressed in response to desiccation.The type of transcript analysis performed in this study sets the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in nematodes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA. adhikaribn@hotmail.com

ABSTRACT

Background: Nematodes are the dominant soil animals in Antarctic Dry Valleys and are capable of surviving desiccation and freezing in an anhydrobiotic state. Genes induced by desiccation stress have been successfully enumerated in nematodes; however we have little knowledge of gene regulation by Antarctic nematodes which can survive multiple environmental stresses. To address this problem we investigated the genetic responses of a nematode species, Plectus murrayi, that is capable of tolerating Antarctic environmental extremes, in particular desiccation and freezing. In this study, we provide the first insight into the desiccation induced transcriptome of an Antarctic nematode through cDNA library construction and suppressive subtractive hybridization.

Results: We obtained 2,486 expressed sequence tags (ESTs) from 2,586 clones derived from the cDNA library of desiccated P. murrayi. The 2,486 ESTs formed 1,387 putative unique transcripts of which 523 (38%) had matches in the model-nematode Caenorhabditis elegans, 107 (7%) in nematodes other than C. elegans, 153 (11%) in non-nematode organisms and 605 (44%) had no significant match to any sequences in the current databases. The 1,387 unique transcripts were functionally classified by using Gene Ontology (GO) hierarchy and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results indicate that the transcriptome contains a group of transcripts from diverse functional areas. The subtractive library of desiccated nematodes showed 80 transcripts differentially expressed during desiccation stress, of which 28% were metabolism related, 19% were involved in environmental information processing, 28% involved in genetic information processing and 21% were novel transcripts. Expression profiling of 14 selected genes by quantitative Real-time PCR showed 9 genes significantly up-regulated, 3 down-regulated and 2 continuously expressed in response to desiccation.

Conclusion: The establishment of a desiccation EST collection for Plectus murrayi, a useful model in assessing the structural, physiological, biochemical and genetic aspects of multiple stress tolerance, is an important step in understanding the genome level response of this nematode to desiccation stress. The type of transcript analysis performed in this study sets the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in nematodes.

Show MeSH
Related in: MedlinePlus