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Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.

Zhong XW, Wang XH, Tan X, Xia QY, Xiang ZH, Zhao P - Int J Mol Sci (2014)

Bottom Line: Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels.The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars.The functions and regulatory mechanism of BmCDA7, however, need further investigation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. zxw_strive@163.com.

ABSTRACT
The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

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Tissue distribution of BmCDA7. (A) The spatial expression profile at the transcriptional level. Total RNA of different tissues from the third day of the fifth instar larvae were used in the RT-PCR analysis. Silkworm actin 3 gene was used as the control; (B) Tissue-specific localization of BmCDA7 protein. Western blotting analysis was performed to detect the expression of the protein. Total protein from different tissues from the third day of the fifth instar larvae were used in this analysis. Tubulin was used as the positive control. PM: Peritrophic membrane; Carcass: All tissues minus alimentary canal.
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f4-ijms-15-01946: Tissue distribution of BmCDA7. (A) The spatial expression profile at the transcriptional level. Total RNA of different tissues from the third day of the fifth instar larvae were used in the RT-PCR analysis. Silkworm actin 3 gene was used as the control; (B) Tissue-specific localization of BmCDA7 protein. Western blotting analysis was performed to detect the expression of the protein. Total protein from different tissues from the third day of the fifth instar larvae were used in this analysis. Tubulin was used as the positive control. PM: Peritrophic membrane; Carcass: All tissues minus alimentary canal.

Mentions: The expression of BmCDA7 was studied by reverse transcription-PCR (RT-PCR; Figure 4A). The foregut, midgut, hindgut and remaining carcass were dissected from larvae on day three of the fifth instar to use in this study. The signal was detected only in the midgut; no signal was found in the foregut, hindgut or remaining carcass. The tissue distribution of BmCDA7 in silkworm larvae was detected by western blot using the antibody to the recombinant protein (Figure 4B). BmCDA7 was detected in the midgut and in PM tissue, but not in the larval foregut, hindgut or remainder of the carcass. This result was confirmed by analysis of the translational levels of BmCDA7. This suggests that the BmCDA7 protein is synthesized in the midgut and transferred to the PM, where it is involved in maintaining the PM molecular structure; BmCDA7 is therefore one of the PM proteins. The expression of the BmCDA7 gene in the newly hatched silkworm larva on day seven of the fifth instar, and a high level of expression in the newly exuviated larvae of different instars, which is the period from formation to the apoptosis of the silkworm midgut, is shown (Figure 5). Therefore, we suggest that the BmCDA7 gene has important roles in updating the PM. Ghormade et al. found the presence of these CDA enzymes in the midgut tissue of larvae only during the feeding period, which might be associated with increased absorption of nutrients [37]. Nahar et al. reported CDA had a significant role in self-defense from the insect chitinases produced during the molting process [38].


Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.

Zhong XW, Wang XH, Tan X, Xia QY, Xiang ZH, Zhao P - Int J Mol Sci (2014)

Tissue distribution of BmCDA7. (A) The spatial expression profile at the transcriptional level. Total RNA of different tissues from the third day of the fifth instar larvae were used in the RT-PCR analysis. Silkworm actin 3 gene was used as the control; (B) Tissue-specific localization of BmCDA7 protein. Western blotting analysis was performed to detect the expression of the protein. Total protein from different tissues from the third day of the fifth instar larvae were used in this analysis. Tubulin was used as the positive control. PM: Peritrophic membrane; Carcass: All tissues minus alimentary canal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958831&req=5

f4-ijms-15-01946: Tissue distribution of BmCDA7. (A) The spatial expression profile at the transcriptional level. Total RNA of different tissues from the third day of the fifth instar larvae were used in the RT-PCR analysis. Silkworm actin 3 gene was used as the control; (B) Tissue-specific localization of BmCDA7 protein. Western blotting analysis was performed to detect the expression of the protein. Total protein from different tissues from the third day of the fifth instar larvae were used in this analysis. Tubulin was used as the positive control. PM: Peritrophic membrane; Carcass: All tissues minus alimentary canal.
Mentions: The expression of BmCDA7 was studied by reverse transcription-PCR (RT-PCR; Figure 4A). The foregut, midgut, hindgut and remaining carcass were dissected from larvae on day three of the fifth instar to use in this study. The signal was detected only in the midgut; no signal was found in the foregut, hindgut or remaining carcass. The tissue distribution of BmCDA7 in silkworm larvae was detected by western blot using the antibody to the recombinant protein (Figure 4B). BmCDA7 was detected in the midgut and in PM tissue, but not in the larval foregut, hindgut or remainder of the carcass. This result was confirmed by analysis of the translational levels of BmCDA7. This suggests that the BmCDA7 protein is synthesized in the midgut and transferred to the PM, where it is involved in maintaining the PM molecular structure; BmCDA7 is therefore one of the PM proteins. The expression of the BmCDA7 gene in the newly hatched silkworm larva on day seven of the fifth instar, and a high level of expression in the newly exuviated larvae of different instars, which is the period from formation to the apoptosis of the silkworm midgut, is shown (Figure 5). Therefore, we suggest that the BmCDA7 gene has important roles in updating the PM. Ghormade et al. found the presence of these CDA enzymes in the midgut tissue of larvae only during the feeding period, which might be associated with increased absorption of nutrients [37]. Nahar et al. reported CDA had a significant role in self-defense from the insect chitinases produced during the molting process [38].

Bottom Line: Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels.The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars.The functions and regulatory mechanism of BmCDA7, however, need further investigation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. zxw_strive@163.com.

ABSTRACT
The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

Show MeSH