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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

Multiple sequence alignment of the core region of the catalytic domains of Ifu-Chit2 and related chitinases. Identical amino acids are high-lighted in yellow, and similar residues are shown in green or light-green. The consensus (average) sequence is shown below the aligned sequences. Highly conserved motifs SxGG and DxxDxDxE are boxed. GenBank accession numbers for are listed in parentheses: A. album CHIT (CAA45468), C. confragosa CHIT2 (AAV98692), I. farinosa CHIT (ABD64606), Lecanicillium fungicola CHIT (AAP45631), Aspergillus fumigatus CHIT (AAO61686), Emericella nidulans CHIT (BAA35140), C. confragosa CHIT1 (AAX56960), I. fumosorosea CHIT1 (FJ377733), Neurospora crassa CHIT1 (EAA36073), Trichoderma virens CHIT (AAL84697), B. bassiana CHIT1 (AY145440), T. harzianum CHIT36Y (AAL01372), Streptomyces avermitilis CHIT (NP_826813), S. coelicolor CHIT (NP_626743), Metarhizium acridum CHIT2 (AJ293217), M. anisopliaeCHIT2 (AAY34347), B. bassiana CHIT2 (AY147011), M. anisopliae CHIT42 (AAB81998), M. flavoviride CHIT (CAB44709), Nomuraea rileyi CHIT (AY264288), Hypocrea rufa CHIT (AAF19617), Stachybotrys elegans CHIT (AAM70478).
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f02: Multiple sequence alignment of the core region of the catalytic domains of Ifu-Chit2 and related chitinases. Identical amino acids are high-lighted in yellow, and similar residues are shown in green or light-green. The consensus (average) sequence is shown below the aligned sequences. Highly conserved motifs SxGG and DxxDxDxE are boxed. GenBank accession numbers for are listed in parentheses: A. album CHIT (CAA45468), C. confragosa CHIT2 (AAV98692), I. farinosa CHIT (ABD64606), Lecanicillium fungicola CHIT (AAP45631), Aspergillus fumigatus CHIT (AAO61686), Emericella nidulans CHIT (BAA35140), C. confragosa CHIT1 (AAX56960), I. fumosorosea CHIT1 (FJ377733), Neurospora crassa CHIT1 (EAA36073), Trichoderma virens CHIT (AAL84697), B. bassiana CHIT1 (AY145440), T. harzianum CHIT36Y (AAL01372), Streptomyces avermitilis CHIT (NP_826813), S. coelicolor CHIT (NP_626743), Metarhizium acridum CHIT2 (AJ293217), M. anisopliaeCHIT2 (AAY34347), B. bassiana CHIT2 (AY147011), M. anisopliae CHIT42 (AAB81998), M. flavoviride CHIT (CAB44709), Nomuraea rileyi CHIT (AY264288), Hypocrea rufa CHIT (AAF19617), Stachybotrys elegans CHIT (AAM70478).

Mentions: The 5′ and 3′ ends of the partial sequence were extended from gene-specific primers using the RACE approach. The 5′-UTR, ORF, and 3′-UTR were 510 bp, 1272 bp, and 202 bp, respectively (Figure 1). The deduced protein was 423 aa with a predicted molecular mass of 46.57 kDa (Figure 1), a predicted isoelectric point (pI) of 5.22, and a 22-aa signal peptide at the N-terminus (shaded, Figure 1). Comparison of the predicted Ifu-Chit2 chitinase with fungal orthologs revealed two highly conserved regions of the catalytic domain (SIGG and DGIDIDWE) that correspond to a substrate-binding site and catalytic residues, respectively. This confirmed that Ifu-Chit2 was a member of glycosyl hydrolase family 18 (Figure 2). A phylogenetic tree showed that Ifu-Chit2 is more closely related to B. bassiana chit2 than to chitinases of other species (Figure 3).


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)

Multiple sequence alignment of the core region of the catalytic domains of Ifu-Chit2 and related chitinases. Identical amino acids are high-lighted in yellow, and similar residues are shown in green or light-green. The consensus (average) sequence is shown below the aligned sequences. Highly conserved motifs SxGG and DxxDxDxE are boxed. GenBank accession numbers for are listed in parentheses: A. album CHIT (CAA45468), C. confragosa CHIT2 (AAV98692), I. farinosa CHIT (ABD64606), Lecanicillium fungicola CHIT (AAP45631), Aspergillus fumigatus CHIT (AAO61686), Emericella nidulans CHIT (BAA35140), C. confragosa CHIT1 (AAX56960), I. fumosorosea CHIT1 (FJ377733), Neurospora crassa CHIT1 (EAA36073), Trichoderma virens CHIT (AAL84697), B. bassiana CHIT1 (AY145440), T. harzianum CHIT36Y (AAL01372), Streptomyces avermitilis CHIT (NP_826813), S. coelicolor CHIT (NP_626743), Metarhizium acridum CHIT2 (AJ293217), M. anisopliaeCHIT2 (AAY34347), B. bassiana CHIT2 (AY147011), M. anisopliae CHIT42 (AAB81998), M. flavoviride CHIT (CAB44709), Nomuraea rileyi CHIT (AY264288), Hypocrea rufa CHIT (AAF19617), Stachybotrys elegans CHIT (AAM70478).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f02: Multiple sequence alignment of the core region of the catalytic domains of Ifu-Chit2 and related chitinases. Identical amino acids are high-lighted in yellow, and similar residues are shown in green or light-green. The consensus (average) sequence is shown below the aligned sequences. Highly conserved motifs SxGG and DxxDxDxE are boxed. GenBank accession numbers for are listed in parentheses: A. album CHIT (CAA45468), C. confragosa CHIT2 (AAV98692), I. farinosa CHIT (ABD64606), Lecanicillium fungicola CHIT (AAP45631), Aspergillus fumigatus CHIT (AAO61686), Emericella nidulans CHIT (BAA35140), C. confragosa CHIT1 (AAX56960), I. fumosorosea CHIT1 (FJ377733), Neurospora crassa CHIT1 (EAA36073), Trichoderma virens CHIT (AAL84697), B. bassiana CHIT1 (AY145440), T. harzianum CHIT36Y (AAL01372), Streptomyces avermitilis CHIT (NP_826813), S. coelicolor CHIT (NP_626743), Metarhizium acridum CHIT2 (AJ293217), M. anisopliaeCHIT2 (AAY34347), B. bassiana CHIT2 (AY147011), M. anisopliae CHIT42 (AAB81998), M. flavoviride CHIT (CAB44709), Nomuraea rileyi CHIT (AY264288), Hypocrea rufa CHIT (AAF19617), Stachybotrys elegans CHIT (AAM70478).
Mentions: The 5′ and 3′ ends of the partial sequence were extended from gene-specific primers using the RACE approach. The 5′-UTR, ORF, and 3′-UTR were 510 bp, 1272 bp, and 202 bp, respectively (Figure 1). The deduced protein was 423 aa with a predicted molecular mass of 46.57 kDa (Figure 1), a predicted isoelectric point (pI) of 5.22, and a 22-aa signal peptide at the N-terminus (shaded, Figure 1). Comparison of the predicted Ifu-Chit2 chitinase with fungal orthologs revealed two highly conserved regions of the catalytic domain (SIGG and DGIDIDWE) that correspond to a substrate-binding site and catalytic residues, respectively. This confirmed that Ifu-Chit2 was a member of glycosyl hydrolase family 18 (Figure 2). A phylogenetic tree showed that Ifu-Chit2 is more closely related to B. bassiana chit2 than to chitinases of other species (Figure 3).

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