A Novel Anti Fungal Peptide From Foxtail Millet Seeds

16

30

Research ArticleReceived: 4 July 2010 Revised: 28 November 2010 Accepted: 25 January 2011 Published online in Wiley Online Library: 28 March 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4359

A novel antifungal peptide from foxtail milletseedsWentao Xu,a,b† Lu Wei,a† Wei Qu,a† Zhihong Liang,a Jinai Wang,a

Xiaoli Peng,b Yanan Zhangb and Kunlun Huanga,b∗

Abstract

BACKGROUND: Antifungal proteins (AFP) help plants to combat phytopathogenic fungi and thus protect plants from thedevastating damage caused by fungal infections and prevent massive economic losses. To date, several proteins withantibacterial and/or antifungal properties have been isolated and characterized from different plant species and tissues;however, there are no reports concerning the antifungal peptide from foxtail millet seeds.

RESULTS: An antifungal peptide with a molecular mass of 26.9 kDa was isolated from dry seeds of the foxtail millet (Setariaitalica (L.) Beauv.), using a procedure that involved four chromatographic steps. The antifungal peptide was adsorbed onCM-Sepharose, Affi-gel blue gel and Superdex 75. It was further purified by C18 reverse-phase high-performance liquidchromatography and submitted for analysis of peptide mass fingerprint. The Mascot peptide mass fingerprint of the isolatedprotein hit no existing protein (score >60), and it was proved to be a novel antifungal peptide. It inhibited mycelial growth inAlternaria alternate with an IC50 of 1.3 µmol L−1, and it also exhibited antifungal activity against Trichoderma viride, Botrytiscinerea and Fusarium oxysporum. Transmission electron microscopy of mold forms of Alternaria alternate after incubation with20 µg mL−1 of the antifungal protein for 48 h revealed marked ultrastructural changes in the fungus.

CONCLUSION: A novel antifungal peptide with high potency was isolated from foxtail millet seeds.c© 2011 Society of Chemical Industry

Keywords: foxtail millet; antifungal peptide; isolation; peptide mass fingerprint; transmission electron microscopy

INTRODUCTIONAntifungal proteins (AFP) help plants to combat phytopathogenicfungi and thus protect plants from the devastating damage causedby fungal infections and prevent massive economic losses. Sys-temic production of the antifungal proteins in crop plants shouldbe taken into account as a novel strategy tos engineer biolog-ical control of fungal pathogens, as shown for AFP and otherantimicrobial proteins.1 – 4 Finally, they could complement or sub-stitute for chemical preservatives in food, as suggested for Sodiumfluoride (molecular formula is NaF).5 Once the active sites ofthese proteins are determined, the activity and specificity rangecould be enhanced by site-directed mutagenesis and/or by thedevelopment of synthetic derivatives, in order to design new com-pounds with pharmaceutical value.6 – 8 Consequently, research onantifungal proteins has attracted the attention of many investiga-tors.

Foxtail millet (Setaria italica (L.) Beauv.) is an important foodcrop gown in India, China and Japan. It is also planted in Australia,North Africa and South America.9 It has long been favored by manylocal farmers for its excellent drought resistance, high toleranceto poor soil and good nutrient value. It has become increasinglypopular and has formed a common dish for many families inrecent years because of its nutritive value.10,11 Minor milletsare nutritionally superior to rice and wheat and the presenceof all the required nutrients in millets makes them suitable for

industrial-scale utilization in the manufacture of foodstuffs (e.g.baby foods, snack foods and dietary food).12,13 However, theproductivity of foxtail millet is limited for various reasons, oneof which is its susceptibility to fungal diseases such as downymildew and ergot, resulting in heavy losses of yield and quality.To date, several proteins with antibacterial and/or antifungalproperties have been isolated and characterized from differentplant species and tissues;14 – 40 however, there are no reportsabout the antifungal peptide from foxtail millet seeds. A detailedanalysis of antifungal peptide that could be effective against thesediseases will therefore be used as an important step towardsthe final objective of enhancing the defense mechanism offoxtail millet. This work will enrich the antimicrobial peptidefamily and may broaden our insight into the plant defensemechanism.

∗ Correspondence to: Kunlun Huang, Laboratory of Food safety, College of FoodScience and Nutritional Engineering, China Agricultural University, Beijing100083, China. E-mail: [email protected]

† These three authors contributed equally.

a Laboratory of Food safety, College of Food Science and Nutritional Engineering,China Agricultural University, Beijing 100083, China

b Supervision and Testing Center of Agricultural Products Quality, Ministry ofAgriculture, Beijing 100083, China

J Sci Food Agric 2011; 91: 1630–1637 www.soci.org c© 2011 Society of Chemical Industry

16

31

A novel antifungal peptide www.soci.org

Figure 1. Ion-exchange chromatography on CM-Sepharose. Column dimensions: 2.5 cm × 25 cm. Sample: the protein was purified by the saturatedammonium sulfate method and filtered using a 0.45 µm membrane filter. Starting buffer: 50 mmol L−1 HAc-NaAc buffer (pH 4.6). Line b across the righthalf of the chromatogram represents 0–1 mol L−1 NaCl concentration gradient used to desorb adsorbed proteins. Antifungal activity was detected infraction CM3 (line a).

Figure 2. Affinity chromatography on an Affi-gel blue gel column. Column dimensions: 5 cm × 10 cm. Sample: fraction CM3 from CM-Sepharose. Startingbuffer: 20 mmol L−1 Tris-HCl buffer (pH 7.4). Line b across the right half of the chromatogram represents 0–1 mol L−1 NaCl concentration gradient usedto desorb adsorbed proteins. Antifungal activity was detected in fraction B3 (line a).

MATERIALS AND METHODSPurification of antifungal peptideFresh foxtail millet seeds (800 g) were extracted with distilledwater using a Waring blender, followed by centrifugation at12 000 × g for 20 min. The resulting supernatant was saved.The proteins were fractionated with ammonium sulfate (AS)by stepwise precipitation, and the concentrations of saturatedAS were selected as 30–60%. The precipitation was dialyzedagainst distilled Water for 24 h at 4 ◦C to remove salinity, andthen the water fraction containing peptides was freeze-driedand stored at −20 ◦C until needed. After homogenization indistilled water (3 mL g−1) and centrifugation (12 000 × g for

20 min), the resulting supernatant was loaded on to a 2.5 cm× 25 cm column of CM-Sepharose (Sigma, St Louis, MO, USA) in50 mmol L−1 HAc-NaAc buffer (pH 4.6). Five peaks were obtained.Fraction CM3 was subjected to affinity chromatography on a 5 cm× 10 cm column of Affi-gel blue gel (Bio-Rad, Sunnyvale, CA,USA) in 20 mmol L−1 Tris-HCl buffer (pH 7.4). The third adsorbedfraction (B3) was subjected to gel filtration on a fast performanceliquid chromatography (PLC) Superdex 75 HR 10/30 column(Amersham Biosciences, Little Chalfont, UK) in 50 mmol L−1

sodium phosphate buffer (pH 7.2) containing 150 mmol L−1 NaClusing an AKTA Purifier (Amersham Biosciences). The first fraction(SU1) represented purified antifungal peptide. High-performance

J Sci Food Agric 2011; 91: 1630–1637 c© 2011 Society of Chemical Industry wileyonlinelibrary.com/jsfa

16

32

www.soci.org W Xu et al.

Figure 3. Gel filtration by fast protein liquid chromatography on aSuperdex 75 HR 10/30 column using an AKTA purifier. Sample: fraction B3from Affi-gel blue gel column. Buffer: 50 mmol L−1 sodium phosphatebuffer (pH 7.2). Flow rate: 0.5 mL min−1. Fraction size: 0.5 mL. Lineb represents 0.15 mol L−1 NaCl concentration. Antifungal activity wasdetected in fraction SU1 (line a).

liquid chromatography (HPLC) with a Kromasil 100-5 C18 column(4.6 mm × 250 mm, 5 µm) was used to further purify the antifungalcompounds (mobile phase: acetonitrile–water = 90/10; flow rate1 mL min−1; temperature 25 ◦C; total retention time 30 min). Theelution was monitored at 220 nm. In order to obtain pure peptidesin quantities sufficient for analysis, several runs were performedand the active fraction was collected.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE), molecular mass determinationSDS-PAGE was conducted according to the method of Laemmliand Favre36 with 5% stacking gel and 15% resolving gel usinga vertical electrophoresis system (Bio-Rad). After electrophoresisthe gel was stained with Coomassie Brilliant Blue. The molecularmass of the antifungal peptide was determined by comparison

of its electrophoretic mobility with those of molecular massmarker proteins from Amersham Biosciences. Matrix-assisted laserdesorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was also employed to determine the molecular massof the purified peptide (autoflex II TOF/TOF, Bruker-Daltonics,Billerica, MA, USA).

Protein microsequencing and peptide mass FingerprintingThe purified antifungal was separated by SDS-PAGE and electro-eluted to a polyvinylidene difluoride (PVDF) membrane. It wasvisualized with Coomassie Blue and submitted to a commercialinstitution for N-terminal sequence assay by Edman degradationwith an Applied Biosystems sequencer (model Procise 491).

For peptide mass fingerprinting, isolation of the protein wasperformed by electrophoresis. The target protein spots wereexcised from the gel directly and then subjected to in-geltrypsin digestion.37 Mass spectrometry was carried out at theCollege of Biological Sciences of China Agricultural Universityon a MALDI-TOF MS (autoflex II TOF/TOF, Bruker-Daltonics).The obtained peak lists were submitted to Matrix Sciences(http://www.matrixscience.com) programmed for protein identi-fication. Additional match of the mascot peptide mass fingerprintof the foxtail millet seeds antifungal peptide with the otherprotein sequence in http://www.ncbi.nlm.nih.gov/blast was alsoperformed (http://www.expasy.org/tools/findpept.html).

Antifungal activity assaysThe antifungal potency of the peptide was examined in differentspecies of fungi, including Trichoderma viride, Alternaria alternate,Botrytis cinerea and Fusarium oxysporum. It was assessed by usingsterile Petri plates (100 mm × 15 mm) containing 10 mL potatodextrose agar. After the mycelial colony had developed, sterileOxford cups were placed at a distance of 0.5 cm from the rimof the mycelial colony. An aseptic solution of the tested samplewas prepared in 50 mmol L−1 sodium phosphate buffer (pH 7.2).An aliquot of a solution of the isolated peptide was added to anOxford cup. The Petri plates were incubated at 26 ◦C for 72 h until

Figure 4. HPLC with a Kromasil 100-5 C18 column (4.6 mm × 250 mm, 5 µm). Mobile phase: acetonitrile–water, 90/10; flow rate, 1 mL min−1; temperature,25 ◦C; total retention time, 30 min. The elution was monitored at 220 nm.

wileyonlinelibrary.com/jsfa c© 2011 Society of Chemical Industry J Sci Food Agric 2011; 91: 1630–1637

16

33


mycelial growth had enveloped the disks containing the controland had produced crescents of inhibition around the disks withantifungal samples.38 – 40

To determine the IC50 value for the antifungal activity, threedoses of the antifungal peptide of foxtail millet seeds were addedseparately to three aliquots each containing 10 mL potato dextroseagar at 45 ◦C, mixed rapidly and poured into three separate smallPetri dishes. After the agar had cooled, a small drop of themycelia suspension was placed in the center of each dish and thenincubated at 26 ◦C for 72 h. When the mycelia of the blank grewand arrived at the edge of the plate, the diameter of the myceliaarea in each dish was measured. From the average diameter ofthree repeated determinations, the doses in µg mL−1 of 50%inhibition of mycelia extension (IC50) were obtained.39,41 – 43

Effect of antifungal peptide on hyphal morphologyTo evaluate the effect of the antifungal peptide on fungal hyphae,we proposed an application using coverslips. Fungi were grown on100 mm × 15 mm Petri plates containing 10 mL potato dextroseagar (PDA) and incubated at 26 ◦C for 72 h. After some Oxford cupswere placed at a distance of 0.5 cm from the rim of the mycelialcolony, blank control and the isolated protein were added andthen coverslips were inserted into the PDA along the directionof the hyphae. After 48 h, hyphal growth and morphology wereexamined and photographed with a light microscope (OlympusBX51T-DP70, Shinjuku-Ku, Tokyo, Japan) at 600× to examinestructural abnormalities.

Transmission electron microscopyIn order to investigate the effect of foxtail millet seed antifungalpeptide on the ultrastructure of Alternaria alternate forms, fungalsamples were examined by transmission electron microscopy.Alternaria alternate treated with the isolated protein (subinhibitoryconcentration) was fixed with 2.5% glutaraldehyde in 0.1 mol L−1

cacodylate buffer (pH 7.2). Subsequently, they were washed in

Table 1. Yields of chromatographic fractions (from 800 g seeds)

Fraction Yield (mg) Recovery (%) Purification (-fold)

Crude extract 3640 100 –

Ammoniumsulfateprecipitation(30–60%)

2973 81.7 1

CM3 222.4 7.5 3.7

B3 65.3 29.4 11.4

SU1 27.5 42.1 42.2

HPLC 26.7 97.1 42.7

cacodylate buffer and postfixed in 1% (w/v) OsO4 in 0.1 mol L−1

cacodylate buffer (pH 7.2) with 1% potassium ferrocyanide and5 mmol L−1 CaCl2 for 30 min at room temperature. The postfixedcells were dehydrated in acetone and embedded in Epon. Ultrathinsections were stained with uranyl acetate and lead citrate, andexamined in a transmission electron microscope (JEM-1230, JEOL,Tokyo, Japan).

RESULTSThe yields of the various chromatographic fractions are presentedin Table 1. Antifungal activity was detected in the fraction of foxtailmillet seeds extract adsorbed on CM-Sepharose (CM3) (Fig. 1) andsubsequently adsorbed on Affi-gel blue gel (B3) (Fig. 2). FractionB3 was fractionated on Superdex 75 into two fractions. Antifungalactivity resided in the first fraction (SU1) (Fig. 3). The active fractionswere purified in the final step by reverse-phase chromatographyon a C18 silica column (Fig. 4) and a single peak of antifungalactivity was obtained. This peak appeared as a single band inSDS-PAGE (Fig. 5(a)). It represented purified antifungal protein.

Figure 5. Identification results of antifungal peptide of foxtail millet seeds by (a) SDS-PAGE and (b) mass spectrometry. (a) Left lane: molecular massstandard from Amersham Biosciences; right lane: purified antifungal peptide (fraction HPLC-1). (b) 100 pmol of the purified peptide was dissolved inwater–methanol (50 : 50, v/v) containing 1% (v/v) acetic acid at a peptide concentration of 5 µmol L−1. The determined molecular mass by spectrometrywas 26898.842.


16

34


Figure 6. Analysis of the antifungal peptide by MALDI-TOF MS.

The purified peptide yielded a single well-resolved peak onmass spectrometry and purity was about 96.8%, calculated usingUNICORN software (Pharmacia & Biotech, Piscataway, New Jersey,USA). Its molecular mass was about 26.9 kDa (Fig. 5(b)). N-terminalsequence assay of the purified peptide was carried out by Edmandegradation, but it could not be sequenced because of the NH2-terminal block.

The antifungal peptide was analyzed further by MALDI-TOF MS(Fig. 6). The Mascot peptide mass fingerprint of this protein hitno existing protein (score >69, http://www.matrixscience.com),and the closest sequence similarity to hypothetical protein (Vitisvinifera, gi 147 865 223) (score, 59) suggested that this peptidemay be a novel peptide. Additional match of the mascot peptidemass fingerprint of the foxtail millet seed antifungal peptide withthe other protein sequence in http://www.ncbi.nlm.nih.gov/wasalso performed (http://www.expasy.org/tools/findpept.html). The

Figure 8. Determination of IC50 value of antifungal activity of an-tifungal peptide of foxtail millet seeds toward Alternaria alternate.(A) Control; (B) 0.6 µmol L−1 antifungal protein; (C) 3 µmol L−1 antifun-gal protein; (D) 15 µmol L−1 antifungal protein. IC50 was determined to be1.3 µmol L−1.

highest sequence similarity (� mass, −0.005 Da) was found indefensin (Setaria italic) (ABM89231), but only 2.36% could bematched.

Antifungal activity of the peptide toward the fungi Trichodermaviride and Alternaria alternate is illustrated in Fig. 7(a) and (b),respectively. Its antifungal activity toward Alternaria alternate withIC50 was determined to be 1.3 µmol L−1 (Fig. 8).

Analysis of hyphal growth and morphology of Alternariaalternate is presented in Fig. 9. The morphological toxicity ofour isolated protein to Alternaria alternate was revealed in mycelialapex offshoot, distortion, tumescence and rupture after treatmentwith 20 µmol L−1 at 26 ◦C for 72 h.

The effect of the antifungal protein on the ultrastructure ofAlternaria alternate is presented in Fig. 10. Analysis of the imagesshowed that control samples had fungal cells surrounded bya specific cell wall composed of dense outer and inner layers

Figure 7. Antifungal activity of purified antifungal peptide of foxtail millet seeds toward (a) Alternaria alternate and (b) Trichoderma viride. (a) (A) Control:100 µL of 50 mmol L−1 PBS buffer (pH 7.2); (B) 20 µmol L−1 antifungal protein; (C) 2.5 µmol L−1 antifungal protein. (b) (A) Control: 15 µL 50 mmol L−1 PBSbuffer (pH 7.2); (B) 50 µmol L−1 antifungal protein; (C) 30 µmol L−1 antifungal protein.


16

35


Figure 9. Microphotographs of Alternaria alternate mycelia grown on PDA with or without antifungal peptide of foxtail millet seeds. (A) Control, normalmycelia of Alternaria alternate; structure is homogenous; (B) control, normal mycelial apex; (C) abnormal mycelia treated with antifungal peptide;(D) abnormal mycelial apex treated with antifungal peptide. The mycelial apex became distorted with the budding of mycelial apex and unusualstructures clearly visible. In addition, some anomalies such as small swellings along the mycelia and mycelial apex were visible; (E)–(H) abnormalmycelia and mycelial apex treated with antifungal peptide, showing mycelia with greater anomalous structures, budded mycelia apex, with cytoplasmicgranulations, mycelia showing clear separation of cytoplasm from cell wall; (I)–(L) abnormal mycelia treated with antifungal peptide, showing cleardecrease in cytoplasmic content. The cytoplasmic reaction and mycelia were damaged.

Figure 10. Transmission electron microscopy of Alternaria alternate fungi forms. Normal ultrastructure of untreated fungi: delimited cell wall composedof dense outer and inner layers separated by a low-density space, and cell with a normal budding profile (A and B) in contrast to the altered morphologyafter treatment with 20 µg mL−1 of the antifungal peptide for 24 h at 37 ◦C: deformed cells, alteration of the space between the cell wall and the plasmamembrane (C, D, F), and irregular budding profile (E). Similar results were obtained in the different analyses.


16

36


separated by a low-density space (Fig. 10(A, B)). Treated cellsshowed an undefined and thickened cell wall, and a change in thespace between the cell wall and the plasma membrane (Fig. 10(C,E, F)). The images showed completely deformed cells, with irregularbudding sites (Fig. 10(D)).

DISCUSSIONTo purify the antifungal peptide, different concentrations ofsaturated ammonium sulfate were selected (30%, 40%, 50%, 60%,70% and 80%). From the SDS-PAGE results and the inhibitoryactivity of antifungal peptide on mycelial growth in Alternariaalternate, we selected the best concentrations, which was therange from 30% to 60%. The antifungal peptide of foxtail milletseeds was adsorbed on CM-Sepharose, Affi-gel blue gel andSuperdex 75. The final purification step was C18 reverse-phaseHPLC. We found that the optimized purification procedure in thispaper could more efficiently gain the antifungal peptide of thefoxtail millet seeds, but it was a little different from the purificationprocedure proven useful for the purification of other antifungalproteins and peptides.20 – 35,44

Although many studies have focused on showing the antifungalactivity of plant derivatives, few have demonstrated their effectson the morphology and ultrastructure of the fungi. The dataobtained in this study showed that Alternaria alternate underwentremarkable alterations, which were visible by light microscope andelectron microscopy when treated with the antifungal peptide.

The hyphae showed lack of cytoplasm, damage and loss ofintegrity and rigidity of the cell wall. The ultrastructural changesincluded thickening of the cell wall, an alteration of the spacebetween the cell wall and the plasma membrane, deformed cells,reduction in cell size and cells with irregular budding sites. Theseobservations indicated that the mode of antifungal activity offoxtail millet antifungal peptide was a result of its attack on the cellwall, retraction of cytoplasm in the hyphae and ultimately death ofthe mycelium. Such modifications may be related to interferenceof the antifungal peptide components by enzymatic reactions ofcell wall synthesis, affecting fungal morphogenesis and growth.

The mechanism of action is not well understood; however, itsdeleterious effect on the cell wall of the fungus may be the mainreason, because the integrity of the cell wall is necessary for celldivision and to allow the expression of molecules involved in theadherence process. Further studies are needed to determine thenature and specific functions of this protein.

In summary, an antifungal peptide with high potency wasisolated from foxtail millet seeds.

ACKNOWLEDGEMENTSThis work was supported by the Ministry of Agriculture and Ministryof Science and Technology of China.

REFERENCES1 Kinal H, Park CM, Berry JO, Koltin Y and Bruenn JA, Processing and

secretion of a virally encoded antifungal toxin in transgenic tobaccoplants: evidence for a Kex2p pathway in plants. Plant Cell 7:677–688(1995).

2 Oldach KH, Becker D and Lorz H, Heterologous expression of genesmediating enhanced fungal resistance in transgenic wheat. MolPlant Microbe Interact 14:832–838 (2001).

3 Osusky M, Zhou G, Osuska L, Hancock RE, Kay WW and Misra S,Transgenic plants expressing cationic peptide chimeras exhibit

broad-spectrum resistance to phytopathogens. Nat Biotechnol18:1162–1166 (2000).

4 Park CM, Berry JO and Bruenn JA, High-level secretion of a virallyencoded anti-fungal toxin in transgenic tobacco plants. Plant MolBiol 30:359–366 (1996).

5 Geisen R, P. nalgiovense carries a gene which is homologous to the pafgene of P. chrysogenum which codes for an antifungal peptide. Int JFood Microbiol 62:95–101 (2000).

6 Epand RF, Umezawa N, Porter EA, Gellman SH and Epand RM,Interactions of the antimicrobial beta-peptide beta-17 withphospholipid vesicles differ from membrane interactions ofmagainins. Eur J Biochem 270:1240–1248 (2003).

7 Hoover DM, Wu Z, Tucker K, Lu W and Lubkowski J, Antimicrobialcharacterization of human beta-defensin 3 derivatives. AntimicrobAgents Chemother 47:2804–2809 (2003).

8 Schaaper WM, Posthuma GA, Plaman HH, Sijtsma L, Fant F,Borremans F, et al, Synthetic peptides derived from thebeta2–beta3 loop of Raphanus sativus antifungal protein 2 thatmimic the active site. J Pept Res 57:409–418 (2001).

9 Sreenivasulu N, Miranda M, Prakash HS, Wobus U and Weschke W,Transcriptome changes in foxtail millet genotypes at high salinity:identification and characterization of a PHGPX gene specifically up-regulated by NaCl in a salt-tolerant line. J Plant Physiol 161:467–477(2004).

10 Ji GS, Du RH, Hou SL, Cheng RH, Wang XY and Zhao XP, Genetics,development, and application of cytoplasmic herbicide resistancein foxtail millet. Agric Sci China 6:779–785 (2007).

11 Veeranagamallaiah G, Chandraobulreddy P, Jyothsnakumari G andSudhakar C, Glutamine synthetase expression and pyrroline-5-carboxylate reductase activity influence proline accumulation intwo cultivars of foxtail millet (Setaria italica L.) with differential saltsensitivity. Environ Exp Bot 60:239–244 (2007).

12 Shinoj S, Viswanathan R, Sajeev MS and Moorthy SN. Gelatinisationand rheological characteristics of minor millet flours. Biosyst Eng95:51–59 (2006).

13 Subramanian S and Viswanathan R, Thermal properties of minor milletgrains and flours. Biosyst Eng 84:289–296 (2003).

14 Chu KT and Ng TB, Isolation of a large thaumatin-like antifungal proteinfrom seeds of the Kweilin chestnut Castanopsis chinensis. BiochemBiophys Res Commun 301:364–370 (2003).

15 Huynh QK, Borgmeyer JR and Zobel JF, Isolation and characterizationof a 22 kDa protein with antifungal properties from maize seeds.Biochem Biophys Res Commun 182:1–5 (1992).

16 Joshi BN, Sainani MN, Bastawade KB, Gupta VS and Ranjekar PK,Cysteine protease inhibit or from pearl millet: a new class ofantifungal protein. Biochem Biophys Res Commun 246:382–387(1998).

17 Lai R, Zheng YT, Shen JH, Liu GJ, Liu H, Lee WH, et al, Antimicrobialpeptides from skin secretions of Chinese red belly toad Bombinamaxima. Peptides 23:427–435 (2002).

18 Ngai PH and Ng TB, Coccinin, an antifungal peptide withantiproliferative and HIV-1 reverse transcriptase inhibitory activitiesfrom large scarlet runner beans. Peptides 25:2063–2068 (2004).

19 Ngai PH, Zhao Z and Ng TB, Agrocybin, an antifungal peptide fromthe edible mushroom Agrocybe cylindracea. Peptides 26:191–196(2005).

20 Parkash A, Ng TB and Tso WW, Isolation and characterization ofluffacylin, a ribosome inactivating peptide with antifungal activityfrom sponge gourd (Luffa cylindrica) seeds. Peptides 23:1019–1024(2002).

21 Wang HX and Ng TB, An antifungal protein from the pea Pisum sativumvar. arvense Poir. Peptides 27:1732–1737 (2006).

22 Wang HX and Ng TB, Ascalin, a new anti-fungal peptide with humanimmunodeficiency virus type 1 reverse transcriptase-inhibitingactivity from shallot bulbs. Peptides 23:1025–1029 (2002).

23 Wang HX and Ng TB, Dendrocin, a distinctive antifungal protein frombamboo shoots. Biochem Biophys Res Commun 307:750–755 (2003).

24 Wang HX and Ng TB, Isolation of cicadin, a novel and potent antifungalpeptide from dried juvenile cicadas. Peptides 23:7–11 (2002).

25 Wang HX and Ng TB, Isolation of cucurmoschin, a novel antifungalpeptide abundant in arginine, glutamate and glycine residues fromblack pumpkin seeds. Peptides 24:969–972 (2003).

26 Wang HX and Ng TB, Isolation of lilin, a novel arginine- and glutamate-rich protein with potent antifungal and mitogenic activities fromlily bulbs. Life Sci 70:1075–1084 (2002).


16

37


27 Wang HX and Ng TB, Purification of allivin, a novel antifungal proteinfrom bulbs of the round-cloved garlic. Life Sci 70:357–365 (2001).

28 Wang HX, Ng TB and Liu Q, Alveolarin, a novel antifungal polypeptidefrom the wild mushroom Polyporus alveolaris. Peptides 25:693–696(2004).

29 Wang SY, Wu JH, Ng TB, Ye XY and Rao PF, A non-specify lipid transferprotein with antifungal and antibacterial activities from the mungbean. Peptides 25:1235–1242 (2004).

30 Wong JH and Ng TB, Vulgarinin, a broad-spectrum antifungal peptidefrom haricot beans (Phaseolus vulgaris). Int J Biochem Cell Biol37:1626–1632 (2005).

31 Xia L and Ng TB, Actinchinin, a novel antifungal protein from the goldkiwi fruit. Peptides 25:1093–1098 (2004).

32 Xia L and Ng TB, Isolation of alliumin, a novel protein with antimicrobialand antiproliferative activities from multiple-cloved garlic bulbs.Peptides 26:177–183 (2005).

33 Ye XY and Ng TB, Isolation of a new cyclophilin-like proteinfrom chickpeas with mitogenic, antifungal and anti-HIV reverse,transcriptase activities. Life Sci 70:1129–1138 (2002).

34 Ye XY, Ng TB and Rao PF, Cicerin and arietin, novel chickpea peptideswith different antifungal potencies. Peptides 23:817–822 (2002).

35 Ye XY, Wang HX and Ng TB, Structurally dissimilar proteins withantiviral and antifungal potency from cowpea (Vigna unguiculata)seeds. Life Sci 67:3199–3207 (2000).

36 Laemmli UK and Favre M, Gel electrophoresis of proteins. J Mol Biol80:573–599 (1973).

37 Shevchenko A, Wilm M, Vorm O and Mann M, Mass spectrometricsequencing of proteins from silver-stained polyacrylamide gels.Anal Chem 68:850–858 (1996).

38 Wong JH and Ng TB, Gymnin, a potent defensin-like antifungalpeptides from the Yunnan bean (Gymnocladus chinensis Baill).Peptides 24:963–968 (2003).

39 Wong JH and Ng TB, Lunatusin, a trypsin-stable antimicrobial peptidefrom lima beans (Phaseolus lunatus L.). Peptides 26:2086–2092(2005).

40 Wong JH and Ng TB, Sesquin, a potent defensin-like antimicrobialpeptide from ground beans with inhibitory activities toward tumorcells and HIV-1 reverse transcriptase. Peptides 26:1120–1126 (2005).

41 Guo YX, Liu QH, Ng TB and Wang HX, Isarfelin, a peptide withantifungal and insecticidal activities from Isaria feline. Peptides26:2374–2391 (2005).

42 Wang HX and Ng TB, An antifungal peptide from red lentil seeds.Peptides 28:547–552 (2007).

43 Wang HX and Ng TB, An antifungal peptide from the coconut. Peptides26:2392–2396 (2005).

44 Chu KT, Xia L and Ng TB, Pleurostrin, an antifungal peptide from theoyster mushroom. Peptides 26:2098–3103 (2005).


Documents

A Novel Anti Fungal Peptide From Foxtail Millet Seeds