cd NA Cloning and sequence analysis of the f ull2length cd NA encoding defensin, an antimicrobial peptide from the housefly ( M usca domestica)

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1 49 (3) : A cta Zoologica S inica 3 cd NA 33 ( ) Cloning and sequence analysis of the f ull2length cd NA encoding defensin an antimicrobial peptide from the housefly ( M usca domestica) WAN G Lai2Cheng WAN G Jin2Xing WAN G Lai2Yuan ZHAO Xiao2Fan ( School of L if e Science S handong U niversity Ji nan Chi na) Abstract Defensin is a kind of cationic inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms. With their broad antimicrobial spectrum and strong pharmaceutical effects antimicrobial peptides including defensins represent a source of novel antibi2 otic agents. A novel full2length 430 base pairs cdna of an insect defensin was cloned using polymerase chain reaction ( PCR) from the cdna library of houseflies ( M usca domestica) that had been challenged by E. coli and S taphylococcus aureus. Sequence analysis revealed that the open reading frame of the cdna encoded a 922amino acid peptide which con2 tained an NH 2 2terminal signal sequence (1-22) followed by a propeptide and the mature peptide (53-92). quence identity with other insect defensin is between 51 % and 73 %. The se2 The mature peptide with a predicted molecular weight of 410 kda and p I of 8169 has 1 negative charged amino acid and 4 positice ones. The putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds Cys12Cys4 Cys22Cys5 and Cys32Cys6. These results suggest that the novel full2length cdna of the defensin gene denominated M dde has been successfully cloned from houseflies [ Acta Zoologica Sinica 49 (3) : ]. Key words Housefly ( M usca domestica) defensin PCR cdna library cdna (Bulet et al. 1999) ( Hoffmann 1995) : (1) ( Hyalophora ce2 ; (2) ( cropia) ) (Drosomysin) ; (3) (Cecropin) ( Hultmark et al. 1980) 500 ( Za2 (Apidaecin) sloff 2002 ) 170 (Abaecins) ( GG ) ( ) ( ) [ This research was funded by grants from the Foundation for Skeleton Staffs of Universities in China ( GG ) Special Foundation for the Doctorship Education ( ) and Foundation of Department of Science and Technolo2 gy of Shandong Province ( ) ] : A Y ( The nucleotide sequence reported in this paper has been submitted to GenBank with ac2 cession number A Y260152) 33 (Corresponding author). E2mail : edu. cn 24 : ν 2003 Acta Zoologica Sinica

2 3 : cdna ( Gloverins) ( Attacins) ( Di2 marcq et al. 1998) ; cdna PCR ( 1999) ( In2 3 5 sect defensins) cdna ( ) 111 (Zasloff 2002) ( M usca domestica) ( S taphylococcus aureus) ; 24 h RNA 112 Taq DNA Pst ( DL2000 DNA Marker Ta KaRa ; UN IQ ) Column DNA Gel Extraction Kit pucm2t Vector ; ( 1992) 113 RNA gt11 cd NA ( 2001 a b) 114 ( 2001) GenBank 5 ( Drosophila) 7 (Rutschmann et al. 2000) MddeF1 MddeR ; ( E. coli ) 115 PCR gt11 cdna MddeF1 MddeR1 PCR : 94 2 min ; s s s 35 ; min PCR : 2 % PCR 116 PCR UN IQ25 Column DNA Gel Extraction Kit (Sangon) PCR pucm2t Vector ( Sangon) PCR pucm2t E. coli DH5 3 Pst Sangon ( )

3 cdna Fig11 Cloning the full2length cdna of defensin from Musca domestica a. PCR 119 bp cdna [cdna fragment (119 bp) of defensin amplified by PCR using primers of MddeF1 and R1 ] b. 3 PCR 253 bp cdna [ Defensin cdna fragment ( 253 bp) including 3 2end untranslated nucleotides amplified by PCR using primers MddeF2 and gtr] primer and specific primer MdeR2 ] NA fragments] c. 5 PCR 352 bp cdna [ 5 2end cdna fragment (352 bp) amplified by PCR using adapter d. 430bp cdna [ Full2length cdna (430 bp) from overlapping of three cd2 M : DL2000 DNA Marker [Lane show amplification results of PCR and lane show the results of negative control of PCR] 117 cd NA 3 5 PCR 119 bp cdna (BLAST) ( http : / / www. ncbi. nlm. nih. gov/ ) 70 % Blastx ( nr) cdna MddeF2 ; ExPASy ( http : / / www. MddeR2 cdna MddeF2 5 GCC ACG TGC GA T TTG TTG A GC 3 MddeR2 5 AC GCA GAC A GC CTT GCC A TT G 3 3 PCR : cdna MddeF2 gt bp gt R PCR 119 bp ( 1a) Blastx : 94 2 min ; s s s 35 ; min 5 ( S arcophaga peregri na) ( Yamada et al. 1994) PCR : cdna 94 % cdna (5 2AA TTCGCGGCCGCT2 3 ) MddeR cd NA PCR Internet NCB I expasy. org/ ) PCR MddeF1 R1 cdna cdna cdna MddeF2

4 3 : cdna cdna Fig12 cd NA and deduced amino acid sequences of defensin from Musca domestica ; ( TAA) Poly (A) (AATAAA) ; ( 3 ) ( TGA) ( TAA) ; [ The sequence of predicted mature peptide is underlined. The Poly ( A) signal ( AATAAA) and the stop codon ( TAA) are boxed. Asterisk ( 3 ) indicates the stop codon ( TGA) followed by the powerful stop codon ( TAA) at the same ORF. The nucleotide sequence is numbered on the left and the amino acid sequence is numbered from the first methionine on the right ] gt11 gt R PCR bp ( 1b) 253 bp 40 3 Poly (A) AA TAAA 3 ; 5 PCR 350 bp ( 1c) 352 bp A TG TAA 5 3 cdna 430 bp cdna ( 1 d) 279 bp kd C1 C4 C2 C5 C3 C6 ( 4) 8144 R A 40 ExPASy Di2 marcq et al. (1998) 3 N loop 1 2 2N loop ; ; kd 8169 ( G) 20 % 6 (C 15 %) 213 cd NA ( Hoffmann cdna ) NCB I Blastx cdna cdna 3 3 : mrna ( 90 % )

5 Fig13 Similarity analysis of deduced defensin amino acid sequence from Musca domestica and other insect defensins 5 - [ Five defensin pre2 cursors (phormicin precursor sapecin precursor defensin 2a defensin precursor of Stomoxys and defensin A precursor of Aedes) from GenBank were compared with defensin precursor of M usca domestica. Shadowed amino acids indicate the identity amino acid ; - was introduced for opti2 mized the alignment of the sequences] 45 ( Ganz et al. 1995) cdna 92 GenBank nestling2sucking blowfly ( Protophorm ia terraenov ae) 4 ( Phormicin precursor ) Fig14 The different structure domains of mature 73 % Phormicin A defensin from Musca domestica Phormicin B 97 % 94 % ( 3) 430 bp cdna 4 : ExPASy (1 22) ( Hoffmann et al. 1999) N 52 (Dimarcq et al. 1998) : (1) ( ) Dimarcq et al. (1998) CS (Drosomycin) ; (2) 2 3 : N loop 2 Thanatin Tachyplesins Androctonin ; 2 2 C 2 (3) ; (4) (C2 C5 C3 C6) N loop (C1 C4) ( Precursor) 19 N ExPASy 38

6 3 : cdna Science 284 : ( References) Bulet P. C. Hetru J. L. Dimarcq and D. A. Hoffmann 1999 Antimicrobial peptides in insects : structure and function. Dev. Com p. I m m unol. 23 (4 5) : Chen L. C. and J. X. Wang 1999 The antibacterial peptides from insects. Progress i n Biotechnology 19 (5) : [ X. F. Zhao 2001 Purification and characterization of an an2 tibacterial peptide from housefly M usca domestica. Journal of S handong U niversity ( Science Edition) 36 (3) : [ ( ) 36 (3) : ] Dimarcq J. L. P. Bulet C. Hetru and J. Hoffmann 1998 Cys2 teine2rich antimicrobial peptides in invertebrates. ( Pepti de Science) 47 : Biopolymers Ganz T. and R. I. Lehrer 1995 Defensins. Pharmac. Ther. 66 : Hoffmann J. A Innate immunity of insect. ion i n I m m unology 7 : Current Opi n2 Hoffmann J. A Immune responsiveness in vector insects. Proc. N atl. Acad. Sci. USA 94 : Hoffmann J. A. F. C. Kafatos C. A. Janeway and R. A. B. Ezekowitz 1999 Phylogenetic perspectives in innate immunity. Hultmark D. H. Steiner T. Rasmuson and H. G. Boman 1980 Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Eur. J. Biochem. 106 : Hyalophora cecropia. Rutschmann S. A. C. J ung R. Zhou N. Silverman J. A. Hoff2 mann and D. Ferrandon 2000 Role of Drosophila IKK in a Toll2independent antibacterial immune response. Nat ure Im2 m unology 1 (4) : Wang J. H. and C. Z. Qu 2000 Progress in insect antimicrobial peptides. Chinese Journal of Vector Biology and Cont rol 11 (5) : [ (5) : ] Wang L. Y. J. X. Wang X. F. Zhao and L. C. Wang 2001a The improved method for the isolation of house fly total RNA. (5) : ] Sichuan J. of Zoology 20 (2) : [ Chen L. C. J. X. Wang Y. Liu L. Y. Wang L. C. Wang and 2001 RNA. 20 (2) : ] Wang L. Y. J. X. Wang X. F. Zhao and L. C. Wang 2001b Construction of cdna library of housefly ( M usca domestica). ological Research 22 (2) : [ 2001 cdna. 22 (2) : ] Wang Y. C. W. Liu and F. Yang 1992 Abstraction of hemolymph of house fly and the inducing of its antibacterial matter. Acta Microbiologica Si nica 32 (6) : [ (6) : ] Yamada K. and S. Natori 1994 Characterization of the antimicro2 Zo2 bial peptide derived from sapecin B an antibacterial protein of S ar2 cophaga peregri na (flesh fly). Biochem. J. 298 : Zasloff M Antimicrobial peptides of multicellular organisms. N at ure 415 :