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Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

Patnaik BB, Kim DH, Oh SH, Song YS, Chanh ND, Kim JS, Jung WJ, Saha AK, Bindroo BB, Han YS - PLoS ONE (2012)

Bottom Line: The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter.The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, South Korea. drbharatbhusan4@gmail.com

ABSTRACT

Background: Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/principal findings: Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/significance: The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

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Full-length sequence of novel gene by 5′- and 3′-RACE-PCR.Total RNA was extracted from M. alba, followed with the generation of 5′- and 3′-RACE ready cDNA The RACE ready cDNAs were amplified using gene-specific primers constructed from the partial cDNA sequence in two PCR reactions (first PCR followed with nested PCR) using universal primer mix. The nested PCR products (both 5′- and 3′-products were cloned in TOPO TA vector and sequenced. (A) 5′-RACE product. (B) 3′-RACE product. Lane-1- 100 bp DNA ladder (Bioneer, Daejeon, Korea); Lane 2- first PCR product with GSP-1; Lane 3- nested PCR product with GSP2.
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pone-0050900-g011: Full-length sequence of novel gene by 5′- and 3′-RACE-PCR.Total RNA was extracted from M. alba, followed with the generation of 5′- and 3′-RACE ready cDNA The RACE ready cDNAs were amplified using gene-specific primers constructed from the partial cDNA sequence in two PCR reactions (first PCR followed with nested PCR) using universal primer mix. The nested PCR products (both 5′- and 3′-products were cloned in TOPO TA vector and sequenced. (A) 5′-RACE product. (B) 3′-RACE product. Lane-1- 100 bp DNA ladder (Bioneer, Daejeon, Korea); Lane 2- first PCR product with GSP-1; Lane 3- nested PCR product with GSP2.

Mentions: The partial cDNA sequence was the basis for the design of primers towards the elucidation of full-length gene sequence by 5′- and 3′-RACE-PCR (Fig. 11). The method used the total RNA extracted from M. alba leaves to construct 5′- and 3′-RACE ready cDNA and two simultaneous PCR reactions (first PCR and nested PCR), followed with cloning into TA vector to get the full-length sequence. The full length sequence has been registered with EBI-ENA under the accession no. HE805964.


Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

Patnaik BB, Kim DH, Oh SH, Song YS, Chanh ND, Kim JS, Jung WJ, Saha AK, Bindroo BB, Han YS - PLoS ONE (2012)

Full-length sequence of novel gene by 5′- and 3′-RACE-PCR.Total RNA was extracted from M. alba, followed with the generation of 5′- and 3′-RACE ready cDNA The RACE ready cDNAs were amplified using gene-specific primers constructed from the partial cDNA sequence in two PCR reactions (first PCR followed with nested PCR) using universal primer mix. The nested PCR products (both 5′- and 3′-products were cloned in TOPO TA vector and sequenced. (A) 5′-RACE product. (B) 3′-RACE product. Lane-1- 100 bp DNA ladder (Bioneer, Daejeon, Korea); Lane 2- first PCR product with GSP-1; Lane 3- nested PCR product with GSP2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526618&req=5

pone-0050900-g011: Full-length sequence of novel gene by 5′- and 3′-RACE-PCR.Total RNA was extracted from M. alba, followed with the generation of 5′- and 3′-RACE ready cDNA The RACE ready cDNAs were amplified using gene-specific primers constructed from the partial cDNA sequence in two PCR reactions (first PCR followed with nested PCR) using universal primer mix. The nested PCR products (both 5′- and 3′-products were cloned in TOPO TA vector and sequenced. (A) 5′-RACE product. (B) 3′-RACE product. Lane-1- 100 bp DNA ladder (Bioneer, Daejeon, Korea); Lane 2- first PCR product with GSP-1; Lane 3- nested PCR product with GSP2.
Mentions: The partial cDNA sequence was the basis for the design of primers towards the elucidation of full-length gene sequence by 5′- and 3′-RACE-PCR (Fig. 11). The method used the total RNA extracted from M. alba leaves to construct 5′- and 3′-RACE ready cDNA and two simultaneous PCR reactions (first PCR and nested PCR), followed with cloning into TA vector to get the full-length sequence. The full length sequence has been registered with EBI-ENA under the accession no. HE805964.

Bottom Line: The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter.The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, South Korea. drbharatbhusan4@gmail.com

ABSTRACT

Background: Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/principal findings: Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/significance: The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

Show MeSH
Related in: MedlinePlus