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Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili.

Futamura N, Totoki Y, Toyoda A, Igasaki T, Nanjo T, Seki M, Sakaki Y, Mari A, Shinozaki K, Shinohara K - BMC Genomics (2008)

Bottom Line: An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology.We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica.Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan. futa@ffpri.affrc.go.jp

ABSTRACT

Background: Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages.

Results: We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes.

Conclusion: Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

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Functional classification and relative levels, as percentages of unique transcripts in the pool, of ESTs derived from male strobili of C. japonica. We assigned 7,369 clusters by reference to databases of KOGs, TWOGs and LSEs using a BLAST-based algorithm (E-value ≤ 10-5). Designations of functional categories: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover and chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; T, signal transduction; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; Y, nuclear structure; Z, cytoskeleton; R, prediction of general function only; S, function unknown; and X, unassigned.
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Figure 1: Functional classification and relative levels, as percentages of unique transcripts in the pool, of ESTs derived from male strobili of C. japonica. We assigned 7,369 clusters by reference to databases of KOGs, TWOGs and LSEs using a BLAST-based algorithm (E-value ≤ 10-5). Designations of functional categories: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover and chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; T, signal transduction; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; Y, nuclear structure; Z, cytoskeleton; R, prediction of general function only; S, function unknown; and X, unassigned.

Mentions: The assembly of ESTs can be expected to generate an overestimate of the actual number of genes represented since failure of ESTs to assemble can result from alternate splicing, differences in usage of polyadenylation sites, sequence polymorphism, and sequencing errors. Levels of redundancy after EST assembly have been estimated to range from 20% to 22% in previous studies of EST collections [23,24]. To reduce redundancy, we compared all consensus sequences (7,686 tentative contigs and 15,972 singletons) using BLASTN after assembly with PHRAP and then we grouped both 5'- and 3'-end sequences derived from the same respective clones together. Finally, our analysis indicated that ESTs derived from 19,437 clones could be grouped into 10,463 clusters as unique transcripts (Table 1; Additional file 1). The largest cluster contained 35 clones, but only 26 transcripts (0.25%) corresponded to more than 10 clones. We found that 6,320 of the 10,463 transcripts (60.4%) corresponded to only one clone and 2,078 clusters (19.9%) corresponded to two clones. The normalization step appeared to reduce the redundancy of our ESTs. Mitochondrial and chloroplast RNA sequences were not filtered, but they contributed one and three clones to the data set of 19,437 cDNA clones, respectively. No contamination by ribosomal RNA was detected in the data set. Figure 1 shows the functional classification of the putative proteins encoded by our ESTs, which was based on assignments in the COG database [25]. Of the putative proteins derived from individual transcripts, 7,339 (70.1%) were assigned to 26 putative functional categories by BLASTX (E-value < 1e-5; Additional file 2).


Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili.

Futamura N, Totoki Y, Toyoda A, Igasaki T, Nanjo T, Seki M, Sakaki Y, Mari A, Shinozaki K, Shinohara K - BMC Genomics (2008)

Functional classification and relative levels, as percentages of unique transcripts in the pool, of ESTs derived from male strobili of C. japonica. We assigned 7,369 clusters by reference to databases of KOGs, TWOGs and LSEs using a BLAST-based algorithm (E-value ≤ 10-5). Designations of functional categories: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover and chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; T, signal transduction; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; Y, nuclear structure; Z, cytoskeleton; R, prediction of general function only; S, function unknown; and X, unassigned.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2568000&req=5

Figure 1: Functional classification and relative levels, as percentages of unique transcripts in the pool, of ESTs derived from male strobili of C. japonica. We assigned 7,369 clusters by reference to databases of KOGs, TWOGs and LSEs using a BLAST-based algorithm (E-value ≤ 10-5). Designations of functional categories: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover and chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; T, signal transduction; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; Y, nuclear structure; Z, cytoskeleton; R, prediction of general function only; S, function unknown; and X, unassigned.
Mentions: The assembly of ESTs can be expected to generate an overestimate of the actual number of genes represented since failure of ESTs to assemble can result from alternate splicing, differences in usage of polyadenylation sites, sequence polymorphism, and sequencing errors. Levels of redundancy after EST assembly have been estimated to range from 20% to 22% in previous studies of EST collections [23,24]. To reduce redundancy, we compared all consensus sequences (7,686 tentative contigs and 15,972 singletons) using BLASTN after assembly with PHRAP and then we grouped both 5'- and 3'-end sequences derived from the same respective clones together. Finally, our analysis indicated that ESTs derived from 19,437 clones could be grouped into 10,463 clusters as unique transcripts (Table 1; Additional file 1). The largest cluster contained 35 clones, but only 26 transcripts (0.25%) corresponded to more than 10 clones. We found that 6,320 of the 10,463 transcripts (60.4%) corresponded to only one clone and 2,078 clusters (19.9%) corresponded to two clones. The normalization step appeared to reduce the redundancy of our ESTs. Mitochondrial and chloroplast RNA sequences were not filtered, but they contributed one and three clones to the data set of 19,437 cDNA clones, respectively. No contamination by ribosomal RNA was detected in the data set. Figure 1 shows the functional classification of the putative proteins encoded by our ESTs, which was based on assignments in the COG database [25]. Of the putative proteins derived from individual transcripts, 7,339 (70.1%) were assigned to 26 putative functional categories by BLASTX (E-value < 1e-5; Additional file 2).

Bottom Line: An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology.We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica.Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan. futa@ffpri.affrc.go.jp

ABSTRACT

Background: Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages.

Results: We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes.

Conclusion: Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

Show MeSH
Related in: MedlinePlus