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Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea.

Meng H, Wang Z, Meng X, Xie L, Huang B - Genet. Mol. Biol. (2015)

Bottom Line: The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18.Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction.The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, China.

ABSTRACT
Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.

No MeSH data available.


Related in: MedlinePlus

Expression analysis of Ifu-chit2 at different times under in vivo conditions. mRNA transcripts were measured at 1, 2, 3 and 4 days after inoculation using qRT-PCR. Relative expression levels were calculated using the comparative ΔΔCt method. The constitutively expressed translation elongation factorgene served as endogenous control, and expression levels are shown as relative to those at 1 day after inoculation, which were given a value of 1. Data are expressed as the mean ± SE (standard error) of three independent experiments. Student’s t-test was used to determine the statistical significance of differences between groups. Differences are considered significant for p < 0.05.
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f06: Expression analysis of Ifu-chit2 at different times under in vivo conditions. mRNA transcripts were measured at 1, 2, 3 and 4 days after inoculation using qRT-PCR. Relative expression levels were calculated using the comparative ΔΔCt method. The constitutively expressed translation elongation factorgene served as endogenous control, and expression levels are shown as relative to those at 1 day after inoculation, which were given a value of 1. Data are expressed as the mean ± SE (standard error) of three independent experiments. Student’s t-test was used to determine the statistical significance of differences between groups. Differences are considered significant for p < 0.05.

Mentions: The expression profiles of Ifu-chit2 under in vivo conditions were investigated using quantitative real-time PCR (Figure 6). A single product-specific melting curve was obtained using for Ifu-chit2, indicating that the primers amplified efficiently and were specific for the gene of interest. Ifu-chit2 gene was found to be expressed constitutively during all stages of insect infection examined (1–4 days). The data normalized to endogenous reference gene were presented as the fold-change in gene expression during different stages of infection and relative to the levels of expression observed at 1 day after infection. Ifu-chit2expression levels peaked at 2 days after infection (Figure 6). Neither chitinase gene was expressed in uninoculated insects.


Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea.

Meng H, Wang Z, Meng X, Xie L, Huang B - Genet. Mol. Biol. (2015)

Expression analysis of Ifu-chit2 at different times under in vivo conditions. mRNA transcripts were measured at 1, 2, 3 and 4 days after inoculation using qRT-PCR. Relative expression levels were calculated using the comparative ΔΔCt method. The constitutively expressed translation elongation factorgene served as endogenous control, and expression levels are shown as relative to those at 1 day after inoculation, which were given a value of 1. Data are expressed as the mean ± SE (standard error) of three independent experiments. Student’s t-test was used to determine the statistical significance of differences between groups. Differences are considered significant for p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612611&req=5

f06: Expression analysis of Ifu-chit2 at different times under in vivo conditions. mRNA transcripts were measured at 1, 2, 3 and 4 days after inoculation using qRT-PCR. Relative expression levels were calculated using the comparative ΔΔCt method. The constitutively expressed translation elongation factorgene served as endogenous control, and expression levels are shown as relative to those at 1 day after inoculation, which were given a value of 1. Data are expressed as the mean ± SE (standard error) of three independent experiments. Student’s t-test was used to determine the statistical significance of differences between groups. Differences are considered significant for p < 0.05.
Mentions: The expression profiles of Ifu-chit2 under in vivo conditions were investigated using quantitative real-time PCR (Figure 6). A single product-specific melting curve was obtained using for Ifu-chit2, indicating that the primers amplified efficiently and were specific for the gene of interest. Ifu-chit2 gene was found to be expressed constitutively during all stages of insect infection examined (1–4 days). The data normalized to endogenous reference gene were presented as the fold-change in gene expression during different stages of infection and relative to the levels of expression observed at 1 day after infection. Ifu-chit2expression levels peaked at 2 days after infection (Figure 6). Neither chitinase gene was expressed in uninoculated insects.

Bottom Line: The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18.Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction.The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, China.

ABSTRACT
Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.

No MeSH data available.


Related in: MedlinePlus