Project description:To identify functions that distinguish the posterior and median cells producing fibroin and sericin in the silk gland of Bombyx mori, serial analysis of gene expression (SAGE) profiles from both silk gland regions were analyzed and compared. The construction of a B. mori reference tag collection extracted from a set of 38000 Bombyx EST sequenced from the 3’ side, helped annotating the SAGE libraries. Most of the tags appeared at similar relative concentration in the two libraries except for those corresponding to silk proteins that were found region-specific and highly abundant. Strikingly, besides tags from silk protein mRNAs, 19 tags were found in the class of high abundance in the median cell library, which were absent in the posterior cell tag collection. Except tags from SP1 mRNA, no PSG specific tags were found in the same class of abundance. The analysis of MSG-specific different transcripts led to suggest that middle silk gland cell realizes more diversified functions as those already known, of synthesis and secretion of the silk sericins.
Project description:To identify functions that distinguish the posterior and median cells producing fibroin and sericin in the silk gland of Bombyx mori, serial analysis of gene expression (SAGE) profiles from both silk gland regions were analyzed and compared. The construction of a B. mori reference tag collection extracted from a set of 38000 Bombyx EST sequenced from the 3’ side, helped annotating the SAGE libraries. Most of the tags appeared at similar relative concentration in the two libraries except for those corresponding to silk proteins that were found region-specific and highly abundant. Strikingly, besides tags from silk protein mRNAs, 19 tags were found in the class of high abundance in the median cell library, which were absent in the posterior cell tag collection. Except tags from SP1 mRNA, no PSG specific tags were found in the same class of abundance. The analysis of MSG-specific different transcripts led to suggest that middle silk gland cell realizes more diversified functions as those already known, of synthesis and secretion of the silk sericins. EST libraries from 11 silkworm tissues were 3’-sequenced to ensure the identification of the most terminal tag. 37,920 sequences were analyzed on ABI 3700 or 3730XL sequencers. Electrophoregrams were processed with KB Basecaller (3730XL traces) or with PHRED (3700 traces) to obtain the .phd files from which were extracted text sequences and their corresponding quality files in Fasta format (Phd2Fasta, Green and Ewing, 1995; 2002). Vector sequences and bad quality regions were removed with an home made software after identification by Lucy (Chou and Holmes, 2001). Chimera were removed by an home made software and retrotransposon sequences were masked by RepeatMasker (http://repeatmasker.org). The cleaned sequences were clusterized with TGICL package (TIGR) and assembled into contigs with CAP3 (Huang and Madan, 1999). Contigs were identified with Blastn and Blastx on GenBank and SwissProt/TREMBL, respectively. Some clusters, splitted by CAP3 procedure, have been regrouped on the basis of Blastx identity. Identitag (Keime et al., 2004) was used to extract all possible tags (forward and reverse) to create a reference database. Moreover, a quality index is attached to each tag depending to the presense of poly-A, polyadenylation signal and its proximity to 3-prime extremity in the original mRNA sequence. This database was supplemented with the tags extracted from public B. mori sequences (GenBank, Silkbase) with the same software. Tags were extracted from MSG and PSG libraries (from 2304 and 3072 sequenced inserts respectively) with the Sagenhaft software (Beissbarth et al., 2004) then identified and compared with Identitag. The assessment of significant differences among the two libraries was performed by using the Z-test used for comparison of SAGE libraries of different size (Kal et al., 1999). For graphic purpose and to avoid division by zero we used a tag value of 1 for tags that were not detected in MSG or PSG libraries. Since the two SAGE libraries showed up different TAG number, we used the Z-test for comparing the two mRNA populations.
Project description:Background: The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production. Results: Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, from the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the genes into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing. Conclusion: The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs in silk production.
Project description:Uric acid (UA) is the final product of purine metabolism and plays an important role as a physiological antioxidant. In recent years, several different groups have reported a correlation between decreased UA in Parkinson’s disease (PD) and clinical progression and stage of PD. However, little is known about the molecular mechanisms of decreased UA under oxidative stress. We used our systematic functional annotation pipeline for silkworm genes to identify a novel UA metabolic pathway regulator under oxidative stress in a UA metabolism mutant silkworm Bombyx mori model. Gene expression was measured in 3day of fifth instar larvae of abnormal uric acid synthesis Bombyx mori mutant of op.
Project description:Uric acid (UA) is the final product of purine metabolism and plays an important role as a physiological antioxidant. In recent years, several different groups have reported a correlation between decreased UA in Parkinson’s disease (PD) and clinical progression and stage of PD. However, little is known about the molecular mechanisms of decreased UA under oxidative stress. We used our systematic functional annotation pipeline for silkworm genes to identify a novel UA metabolic pathway regulator under oxidative stress in a UA metabolism mutant silkworm Bombyx mori model.
Project description:Background: The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production. Results: Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, from the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the genes into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing. Conclusion: The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs in silk production. Sequencing 10 total RNA samples from the posterior silk gland of different strains and developmental stage using Illumina Solexa technology. Four strains of silkworm (Q, B, QB and BQ) with different two development stages (stage 1: fourth instar molting to day 2 of fifth instar; stage 2: fifth instar day 3 to day 8 before spinning, according to our previous genes expression cluster analysis), and two strains (R1 and J1) from entire period (stage 1 + stage 2).