title: Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.
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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, South Korea. drbharatbhusan4@gmail.com
authors.forename: Bharat Bhusan
authors.initials: BB
authors.lastname: Patnaik
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Seung Han
authors.initials: SH
authors.lastname: Oh
authors.forename: Yong-Su
authors.initials: YS
authors.lastname: Song
authors.forename: Nguyen Dang Minh
authors.initials: ND
authors.lastname: Chanh
authors.forename: Jong Sun
authors.initials: JS
authors.lastname: Kim
authors.forename: Woo-jin
authors.initials: WJ
authors.lastname: Jung
authors.forename: Atul Kumar
authors.initials: AK
authors.lastname: Saha
authors.forename: Bharat Bhushan
authors.initials: BB
authors.lastname: Bindroo
authors.forename: Yeon Soo
authors.initials: YS
authors.lastname: Han
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001483:Base Sequence; D012831:Bombyx / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004063:Digestion; D004064:Digestive System; D005243:Feces / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D031625:Morus / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D017421:Sequence Analysis; D014780:Viruses / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D000998:Antiviral Agents; D018076:DNA, Complementary; D006023:Glycoproteins; D010940:Plant Proteins; C058814:germin