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Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus.

Obermeier C, Hosseini B, Friedt W, Snowdon R - BMC Genomics (2009)

Bottom Line: The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant.Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Justus Liebig University Giessen, Department of Plant Breeding, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. christian.obermeier@agrar.uni-giessen.de

ABSTRACT

Background: Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.

Results: Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development.

Conclusion: This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species.

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Real-time RT-PCR expression profile during B. napus seed development using primers specific for the coding (A – sense transcripts) and non-coding strands (B – antisense transcripts) of a group of napin and napin-related transcripts. Square symbols represent napin transcripts most similar to 2S seed storage proteins AT4G27140, AT4G27150, AT4G27160, AT4G27170, while triangular symbols represent napin-related transcripts most similar to protease inhibitor/seed storage/lipid transfer protein AT5G54740.
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Figure 3: Real-time RT-PCR expression profile during B. napus seed development using primers specific for the coding (A – sense transcripts) and non-coding strands (B – antisense transcripts) of a group of napin and napin-related transcripts. Square symbols represent napin transcripts most similar to 2S seed storage proteins AT4G27140, AT4G27150, AT4G27160, AT4G27170, while triangular symbols represent napin-related transcripts most similar to protease inhibitor/seed storage/lipid transfer protein AT5G54740.

Mentions: For comparison of SAGE results with quantitative Real-time RT-PCR detection, two sets of primers and their antisense reverse-complemented sequences were derived from SAGE primer amplified napin 3'-termini (see above) and from assembled contigs of 8,977 B. napus ESTs aligning with an e-value cut-off of 1e-6 to A. thaliana 2S seed storage protein 1 (AT4G27140). One primer set (napin) was designed specifically for the major cluster of B. napus assembled EST contigs most similar to the A. thaliana 2S seed storage proteins 1–4 (59% to 70% on the aa level). Another set of primers (napin-related) was designed specifically for the minor cluster of B. napus assembled EST clusters grouping in-between the four A. thaliana 2S seed storage proteins and the related protease inhibitor/seed storage/lipid transfer (LTP) family protein AT5G54740 (about 47% to 57% identity on the aa level). AT5G54740 represents the next closely related protease inhibitor/seed storage/lipid transfer protein (LTP) family protein gene in A. thaliana, which like other more distantly related proteins of this large family (about 113 members in A. thaliana) exhibits an AAI_SS domain and shares about 53–57% aa identity with the A. thaliana 2S seed storage proteins 1–4. Figure 3 shows the expression profile from days 17 to 70 after pollination from seeds for sense and antisense napin and napin-related transcript expression. The coexpression of sense and antisense transcripts was confirmed for a minor subgroup of napin-related transcripts by strand-specific Real-time RT-PCR, but not for the major napin transcript group as indicated by LongSAGE.


Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus.

Obermeier C, Hosseini B, Friedt W, Snowdon R - BMC Genomics (2009)

Real-time RT-PCR expression profile during B. napus seed development using primers specific for the coding (A – sense transcripts) and non-coding strands (B – antisense transcripts) of a group of napin and napin-related transcripts. Square symbols represent napin transcripts most similar to 2S seed storage proteins AT4G27140, AT4G27150, AT4G27160, AT4G27170, while triangular symbols represent napin-related transcripts most similar to protease inhibitor/seed storage/lipid transfer protein AT5G54740.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719671&req=5

Figure 3: Real-time RT-PCR expression profile during B. napus seed development using primers specific for the coding (A – sense transcripts) and non-coding strands (B – antisense transcripts) of a group of napin and napin-related transcripts. Square symbols represent napin transcripts most similar to 2S seed storage proteins AT4G27140, AT4G27150, AT4G27160, AT4G27170, while triangular symbols represent napin-related transcripts most similar to protease inhibitor/seed storage/lipid transfer protein AT5G54740.
Mentions: For comparison of SAGE results with quantitative Real-time RT-PCR detection, two sets of primers and their antisense reverse-complemented sequences were derived from SAGE primer amplified napin 3'-termini (see above) and from assembled contigs of 8,977 B. napus ESTs aligning with an e-value cut-off of 1e-6 to A. thaliana 2S seed storage protein 1 (AT4G27140). One primer set (napin) was designed specifically for the major cluster of B. napus assembled EST contigs most similar to the A. thaliana 2S seed storage proteins 1–4 (59% to 70% on the aa level). Another set of primers (napin-related) was designed specifically for the minor cluster of B. napus assembled EST clusters grouping in-between the four A. thaliana 2S seed storage proteins and the related protease inhibitor/seed storage/lipid transfer (LTP) family protein AT5G54740 (about 47% to 57% identity on the aa level). AT5G54740 represents the next closely related protease inhibitor/seed storage/lipid transfer protein (LTP) family protein gene in A. thaliana, which like other more distantly related proteins of this large family (about 113 members in A. thaliana) exhibits an AAI_SS domain and shares about 53–57% aa identity with the A. thaliana 2S seed storage proteins 1–4. Figure 3 shows the expression profile from days 17 to 70 after pollination from seeds for sense and antisense napin and napin-related transcript expression. The coexpression of sense and antisense transcripts was confirmed for a minor subgroup of napin-related transcripts by strand-specific Real-time RT-PCR, but not for the major napin transcript group as indicated by LongSAGE.

Bottom Line: The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant.Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Justus Liebig University Giessen, Department of Plant Breeding, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. christian.obermeier@agrar.uni-giessen.de

ABSTRACT

Background: Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana.

Results: Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development.

Conclusion: This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species.

Show MeSH
Related in: MedlinePlus