Tap chi Khoa hpc vS Cdng nghe 49 (1) (2011) 93-99
PHAN TiCH TRiNH TU GIEN PHES CHO VIEC XAC DINH LOAI VI
K H U A N L A C T I CSINH BACTERIOCIN
Nguyen Thj Lam Doan\ Ngo Xuan Manh\ Nguyen Thi Da^
Vu Thj Hang\ Nguyen Xuan Bac^
Khoa Cong nghe thuc pham, Trudng Dgi hoc Nong nghiep Ha Noi 'Vien Cdng nghe sinh hgc, Vien Khoa hoc vd Cong nghe Viet Nam
Lien he vdi tdc gia: nlddoaniayahoo.com Den Tda soan ngay
I.MODAU
Bacterioein cua vi khudn lactic Id mdt trong nhirng hudng da vd dang dupe quan tam ciia cong nghe sinh hgc hien dai. Cdc bacterioein eiia vi khudn dugc xem Id eo ti^m nang to Idn ttong bao qudn vi hau bet vi khudn lactic dugc coi Id an todn. Bacterioein cua vi khudn lactic dugc coi Id nhan td lic che ty nhien, ngan ngira nhirng vi khudn gay benh vd gay thdi hdng frong thuc pham [3,4]. Vi vay ngdy cang cd nhieu phdt hien ve bacterioein mdi do vi khuan lactic sinh ra [1, 11,]. Chinh vi vay, ttong nghien cuu ndy dinh ten lodi cua chung sinh bacterioein cao de budc dau cd the biet bacterioein dugc sinh ra tir lodi nao thudc nhdm nao. Tmdc day phan loai vi sinh vat theo cdc phuang phdp tmyen thdng dya cdc dac tinh hinh thai, sinh li vd hda sinh ....
[12] Cdc dac trung ndy ddi khi ciing bpc Id nhirng ban che dan den nhieu trudng hgp phai xdc dinh lai ten phan loai cua mdt sd vi sinh vat. Tir nhirng ban che tren cimg vdi nhirng tien bd ttong sinh hpc phan tu da md ra khd nang ung dung hixu hieu trong phan loai hpc vd nghien cuu da dang vi sinh vat. Neu nhu cdc phuang phdp tmyen thdng chi cd the tap tmng tten nhiJng ddi tugng vi sinh vat nudi cdy dugc thi phuang phdp sinh hpc phan tu ed the dp dung ddi vdi ed nhirng vi sinh vat nudi cdy dugc vd nhung vi sinh vat khong nudi cay dugc [12]. Dac biet vi khudn lactic Id mdt nhdm rdt nhi6u chi vd ngay trong mdt chi ciing co sy da dang rdt Idn we lodi nen thudng khd khi su dung phuang phdp tmy§n thdng d6 xdc dinh chiing ngay cd d miic chi. Vi vay, nghien cuu ndy tap tmng vdo ung dung kT thuat sinh hpc phan fix dya tren phdn tich dir lieu trinh ty gien pheS dk xdc dinh chiing vi khudn lactic sinh bacterioein cao tir nem chua. Trong thdi gian gdn day, mdt doan gien pheS ngdn rdt dugc quan tam nhu mpt irng cu vien ti6m nang dl xdc dinh mdi quan he phdt sinh lodi. Gien pheS ma hod cho protein phenylalanyl - tRNA synthase, day Id protein ddng vai trd Id enzini cd tdc dung xiic tdc cho viec chuyin phenylalanine d6n tRNA dl tham gia vdo qua trinh sinh tdng hop protein [2,7]. Da cd nhilu nghien cuu cho thdy rdng sir dung doan gien ngdn ma hda cho mdt protein ndo dd nhu mpt dung cu hiiu hieu de xdc dinh cdc lodi ban la sir dung gien ribosomal vi chiing cho ti le khdc nhau Idn vd cho phep sy phan biet cao cua cdc loai gdn nhau trong nhdm vi khudn lactic ban hau het xdc dinh nhd phan tich ttat tu gien 16S rRNA [5, 6, 8]. Sdn phdm PCR la mdt doan gien dai 450 base pair [9]. Day la cong cu rdt cd gid tri tiong phan tich ngudn gdc phdt sinh lodi cua cdc chiing.
Nguyin Thj LSm DoSn, Ngd XuSn M^nh, Nguyin Thj DS.VQ Thj HSng, Nguyin XuSn BSc
2. VAT LIEU vA PHUON(; P H A P NGHIEN ClTU 2.1. Vat lifu nghien cu'u
Chiing vi khudn lactic sinh bacterioein cao dd dugc phdn lap tir mau nem chua Hd Ndi (ki hieu chiing DV4.10). (hung DV4.10 thupc gram (U, ll bdo hinh cdu, khdng cd boat tinh catalase, co boat tinh sinh long hgp bacterioein.
2.2. Phuotig phap nghien cu-u 2.2.1. Phmrng phd/> idch chici DNA
Chiing sinh bacterioein manh nay dugc nudi cay trong mdi tmomg MRS agar (Oxoid) tai 3 0 T trong 24 gid. Chung nay dugc ma hda R-42380 (DV4.1()). DNA genomic ciia chiing dugc tach chilt theo phuang phap eua Cievers et al. 2001 [5]. Sau khi nudi cdy dugc 24 gid, sinh khIi ll bao dugc chuyen vao 6ng eppendorf va dugc rira vdi 800 pl J i;S. Li tdm 10 phiit tdc dp 5000 vdng trong 1 phut o 4"C , loai bd phan dung djch thu sinh khdi tc bdo dc bdo quan trong tii lanh 2 0 T (t6i thilu I gid - toi da 1 tuan). Tilp theo tiln bdnh tach ehilt DNA theo cdc budc sau:
Rira tl bao (Idm tan lanh - rua tl bdo vdi 400 pl TES - li tdm 5 phut tdc dp 1000 vdng trong 1 phut d 4"C - loai bd phan dung dich).
Phd vd tl bdo (cho 300 pl STET vao eppendorf cd chira te bdo d tren - Them 75 pl lysozyme - mutanolysine - Qt 3TC trong 60 phut - Cho 40 pl SDS 20",. dm d 37°C Them
10 mg edt trdng - Lde manh 60 giay - Gid d 37"C trong 10 phut Tiep tuc giii d 65°C ttong 10 phiit).
Tinh sach dung dich DNA: them 100 pl dung djch dem TE - cho 515 pl hon hgp dung dich phenol: chloroform: isoalmylalcohol thep li le 24 : 24 : I - li tdm 5 phiit tdc do 13000 vong trong 1 phiit d 4 °C - chuyen phdn djch ndi sang dng eppendorf mdi.
Ket tiia DNA: cho 70 pl NaCl 5M vd I ml isopropanol lanh - ddo nguge eppendorf 1 2 lan de tua DNA, li tdm 30 phut tdc dp 13000 vdng trong I phiit a 4 ''C - dd bd djch ndi mdt edch nhe nhdng
Rira vd Idm khd DNA: cho 500 pl ethanol 70" o lanh li tdm 5 phiit tdc dp 13000 vdng trong 1 phiit a 4°C dd bd djch ndi mdt edch nhe nhdng 1dm khd tren giav tham, de khd ty nhien trong khdng khi khodng 20 phut - them 100 - 150 pl dung djch TE - bdo qudn DNA d - 20°C - ngdy tiep theo cho them 1,5 pl RNAase 10 mg/ml va ii a 37"C trong 1 gid.
Chdt lugng vd dp tinh sach DNA dugc kiem tra bdng edch do quang phd d budc sdng 234,' 260 vd 280 nm (SpectraMax Plus384, Molecular Devices, Califomia, USA) vd dien di tten gel 1% w/v agarose trong 45 phiit a dien the 75V ciia 5 pL DNA ttdn vdi 2 pL loading dye (4g sucrose vd 2,5 mg bromophenol blue hda tan trong 6 mL dpm TE). Marker dugc su dung la SmartLadder, Eurogentec, Searing, cua Bi.
2.2.2. Phuorng phdp khuech dgi PCR cho gidi trinh tu gien pheS
Mdi PheS-21-F (5'-CAYCCNGCHCGYGAYATGC-3') vd PheS-22-R (5'- CCWARVCCRAARGCAAARCC-3') dugc dimg d l nhan len doan gien pheS [6]. Phdn img dugc tiln hanh vdi DNA pha loang (OD=l). The tich PCR cua 50 pl gdm 5 pl cua lOx PCR buffer (15 mM MgCl2), 5 pl deoxynucleoside triphosphates (2 mM cua mdi loai dNTP), 0,5 pL ciia mdi mdi (50 pM), 1 pL Taq polymerase (lU pl"'), 37 pL nude edt vd 1 pl ciia dung dich
PhSn tich trinh twqien Phes cho viec xSc djnh loai vi khuan lactic sinh bacterioein
DNA. PCR dugc thyc hien sii dung mdy GeneAmp PCR 9600 thermocyeler (Applied Biosystem). Chu trinh nhiet bao gdm (1) 5 phut d 95T, (2) 3 chu trinh 1 phut d 95"C + 2 phut 15 giay d 46°C + 1 phut 15 giay d 72 °C, 30 ehu trinh 35 giay d 95"C + 1 phut 15 gidy d 46"C + 1 phut 15 gidy d 72°C, (4) cudi ciing 7 phut 72"C [6]. Sdn phdm PCR dugc kilm tra bdng edch lay 5 pl sdn phdm PCR vdi 2 pl cua loading dye, dien di tren gel agarose 1% d 75 V trong 45 phut. Marker dugc su dung Id SmartLadder, Eurogentec, Searing, ciia Bi. Sau'khi kilm tra nlu san phdm PCR cd kich thudc dung nhu kich thudc cua doan gien pheS se dugc tinh sach nhd sir dung he thdng mdng loc Nucleofast 96 PCR (Maeherey Nagel). Tilp theo do dl gidi trinh tu gien pheS thi sdn phdm PCR \ ira tinh sach d tren se dugc chay PCR Idn thir hai. Thl tich PCR Id 10 pl gdm 3 pl sdn pham PCR da dugc tinh s^ch, 1.857 pl nude edt, 1,857 pl 5 x dung djch dem sequencing, mdi 3 pl (4 pM) mdi xudi vd moi nguge tach biet, Bigdye 0,286 pl. Chuang trinh nhiet gom 30 chu trinh 15 gidy d 96"C + 1 gidy a 35"C + 4 phiil d 60T. Sau do 10 pl sdn phdm PCR cho mdi xudi \a 10 pl sdn phdm PCR cho mdi nguge dugc chuyin vdo 2 gilng ciia dTa 96 gieng vd them 45 pl sung djch SAM, 10 pl XTerm. Gidi trinh ty dugc thyc hien nhd mdy ABI PRISM 3100 (Applied Biosystem).
2.2.3. Phuang phdp phdn tich trinh tu gien pheS vd .xdc dinh lodi
Sd lieu trinh ty thd dugc chuyen den AutoAssemble software 1.40 (applied Biosystem) hoac GeneBuilder (Applied Maths), trinh ty lien tuc eiia gien PheS dugc xdc djnh. Trinh tu lien tuc ndy se dugc nhap vdo chuang trinh Bionumerie 5.1 (Applied Maths), gid tri tuang dong cua cdc lodi \a cay phdn loai dugc tao ra dya vdo phucmg phdp neighbourjoining vd maximum- parsimony [10]. Trinh ty ciia chiing ndy dugc so sdnh vdi cdc lodi trong ngdn hdng gien BCCM/LMG ciia Dai hpc Gent (Gent, Bi) hoac chudi DNA ndy dugc so sdnh vdi ngdn hdng trinh ty nueleotit qudc te (EMBL) vd thyc hien nhd sir dung chuang trinh FASTA de xdc dinh lodi CO mdi lien quan gdn nhdt da biet eiia trinh ty ttong phdn gien pheS.
3. KET QUA NGHIEN CUtJ vA T H A O LUAN 3.1. Chdt lugng va do tinh sach DNA cua chiing DV4.10
10000 b
M. Marker 1. DV4.10
Hinh 1. Chat lugng va dp tinh sach DNA cua chung DV4.10 tren gel agarose 1%
DNA genomic cua chung DV4.1:0 dugc tdch chilt theo phuang phdp eua Gevers et al. 2001 [5] nhu protocol ttong phdn 2.1. Chdt lugng va dp tinh sach DNA dugc kiem tta bang edch dien di tten 1% w/v agarose ttong 45 phiit d dien thl 75V cua 5 pL DNA trdn vdi 2 pL loadmg dye.
Ket qud dugc chi ra trong hinh 2.
Nguyin Thj LSm DoSn, Ngd XuSn M?nh, Nguyin Thj DS, VQ Thj HSng, Nguyin XuSn BSc
Hinh I cho Ihdy sau khi chay diC-n di mau DNA genomic ciia chiing DV4.10 chi thu mpt vach dam co kich Ihude Idn ban 10000 bp. Chung to tdch chilt DNA eiia chiing DV4.10 dd thanh cdng, khdng co hicn livng dirl gay DNA vd khdng lan RNA.
3.2. San pham PCR doan 0cn pheS ciia chiing DV4.10
Sau khi DNA ciia chiing DV4.I0 tach chill, lien hanh nhdn doan gien/J/JC^ vdi cdc primer va protocol nhu phdn 2.2. San pham PCR dugc chay dien di tren gel agarose 1% Ket qud dien di sdn phdm PCR da thu dugc mpt bang khodng 450 bp (hinh 2) tuang ung vdi kich thudc cua doan gien PheS [9]. Nhu vay, khulch dai doan gien PheS cua chung DV4.10 da cho kit qud tit.
450 b p ^
M Marker I. DV4.10
400 bp - 200 bp
Hinh 2. San pham PCR doan gien p/?eS ciia DV4.10 tren gel agarose 1%
3.3. Giai trinh tu DNA ciia chiing DV4.10 va xdc djnh loai
Bdng 1. Xdc djnh lodi ciia chiing DV4.10 nhd so sdnh \ di ngdn hang trdt ty nucleotide (EMBL)
Chiing
DV4.10
Loai c6 moi quan h^ gan nhat Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis
subsp.cremoris Lactococcus lactis
subsp.cremoris ..
%ID 98 97,8 92,4
92,2
Accession no.
EM_PRO:AE005176 EM_PRO:CP001834 EM_PRO:CP000425 EM_PRO:AM406671
PhSn tich trinh twqien Phes cho viec xac dinh loai vi khuan lactic sinh bacterioein
75.4 79-1
81,4
7 4 5
100 99 5
Lactobacillus nagelii Lactobacillus salivarius Pediococcus pentosaceus Pediococcus acldilactici Lactobacillus plantarum
Streptococcus dysgalactiae subsp. equis.
Streptococcus phocae Enterococcus aquimarinus Enterococcus aquimarinus Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis - Lactococcus lactis subsp. lactis
- Lactococcus lactis subsp. cremoris
LMG 21593T LMG 9476 LMG 10478 LMG 13369 LMG 18027 LMG 15851 LMG 16736 LMG 16612 LMG 16607T LMG 7930 LMG 8516 LMG 9471 LMG 6890T
•'R-42380 (DV- .10 LMG 8505
Hinh 3. Cay phat sinh chiing loai cua DV4.10 duae so sanh vdi cac loai trong ngan han^ gien pheS cua BCCM LMG cua Dai hpc Gent (Gent, Bi), so Bootstrap (> 50), thudc do thl hien
10 nueleotit khac nhau tren 100 nucleotide so sanh
Sau khi kiem tta neu sdn phdm PCR cd kich thudc dung nhu kich thudc ciia doan gien pheS se dupe tinh sach chay PCR vdi mdi xudi, mdi nguge tdch biet vd sir dung chuang trinh nhiet nhu da mieu td frong phdn 2.2. Trinh ty eua chiing ndy dugc so sdnh vdi cdc lodi trong ngan hdng gien BCCM/LMG cua Dai hgc Gent (Gent, Bi) hoac doan DNA ndy dugc so sdnh vdi ngdn hdng trinh ty nueleotit qudc te (EMBL) vd thyc hien nhd sii dung chuang trinh FASTA dk xac djnh lodi cd mdi lien quan gdn nhdt da biet ciia trinh ty trong phdn gien pheS. Ket qud dugc frinh bdy d bdng 1 vd hinh 3.
Nhu vay, khi so sdnh trinh ty gien pheS ciia chiing DV4.10 vdi ngdn hdng trinh ty nucleotide qudc tl (EMBL) nhd sir dung chuang trinh FASTA thi chung ndy eo mdi lien quan gdn nhat vdi lodi Lactococcus lactis subsp. lactis gidng nhau ve trinh ty len 98% sau dd den gilng vdi lodi Lactococcus lactis subsp.cremoris Id 92,4% (bdng 1). Nhu vay, chiing ndy Id lodi Lactococcus lactis subsp. lactis. Kit qud ndy ciing tuang ddng vdi kit qud khi so sdnh chung ndy vdi ngdn hdng gien pheS cua cdc lodi vi khudn trong ngdn hdng gien ciia BCCM/LMG ciia Dai hpc Gent (Gent, Bi). Khi dua din ngdn hdng gien cua BCCM/LMG ciia Dai hpc Gent, Bi thi eliung DV4.10 dugc nhdn mdt so R-42380 (DV4.10). 6 ngdn hdng gien ndy chung DV4.10 co trinh ty ttrang ddng vdi chiing LMG7930, LMG8516, LMG9471, LMG6890T cae chung nay diu Id lodi Lactococcus lactis subsp. lactis. Sy ttrang ddng vl trinh ty gien PheS cua chung DV4.10 vdi cdc chung nay la 97,4%. Trong khi dd chung DV4.10 co trinh ttr ttrang ddng 92,6%
vdi LMG 8505 (Lactococcus lactis suhsp.cremoris) (hinh 3).
Qua cdc kit qud tren thi chung DV4.10 Id lodi Lactococcus lactis subsp. lactis.
4. KET LUAN
Bdng kl thuat phan tich trinh tu gien pheS, da xdc dinh chung (DV4.10) cd boat tinh sinh ting hgp bacterioein manh Id lodi Lactococcus lactis subsp. lactis. Nhu vay, vdi viec su dung doan gien ngdn ma hda cho mot protein ndo dd, gien pheS ma hod cho protein phenylalanyl -
Nguyin Thj LSm DoSn, Ngd XuSn M^nh, Nguyin Thj DS, VQ Thj HSng, Nguyin XuSn Bic
tRNA synthase nhu mpt dyng cy hiru hipu dl xac djnh cdc lodi vd cho ph^p phdn biet cao giua cdc lodi giin nhau Irong nhom vi khuan lactic.
Lai cSm an. T^c gi^ xin dugc c^m an si/ hgp t^c cija phdng thi nghiem Vi sinh v§t cua trudng Dgi hpc Ghent, Bf tgo dieu ki^n d^ t^c g\S thyc hi$n ndi dung nghidn ciru n^y v^ c^m an t^t ca c^c dong nghiep.
T A l L l f U T H A M K H A O
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Chobert, and T. Haertle Purillealion and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. Journal of Appl. Microbiology 101 (2006) 837-848.
2. D. Beyer, H. P Kroll, R. Endermann, G. Schiffer, S. Siegcl, M. Bauser, J. Pohlmann, M.
Brands, K. Ziegelbauer, D. Haebich, C. Eymann, and H. B. Oesterhelt New Class of Bacterial Phenylalanyl-lRNA Synthetase Inhibitors with High Potency and Broad- Spectmm Activity, Antimimicrobial agents and chemotherapy (2004) 525-532.
3. E. C. M. Bruno and T. J. Montville Common Mechanistic Action of Bacteriocins from Lactic acid Bacteria, Appl Environ Microbiol 59 (1993) 3003-3010.
4. P M. Davidson, J. N. Sofos, A. L. Brane Antimicrobial in food. Third edition, CRC Press Toylaf and Francis group.
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6. D. B. Katrien, C. M. A. P Franz, M. Vancanneyt, U. Schillinger. F Mozzi, G. F D.
Valdez, L. D. Vuyst, and P Vandammel Pediococcus argentinicus sp. nov. from Argentinean fermented wheat flour and identification of Pediococcus species by pheS, rpoA and atpA sequence analysis, Intemational Joumal of S\ stematic and Evolutionary Microbiology 58 (2008) 2909-2916.
7. D. B. Katrien, N. Camu, L. D. Vuyst, and P Vandamme Laciobacillus fabijermentans sp. nov. and Lactobacillus cacaonum sp. nov., isolated from Ghanaian cocoa fermentations, Intemational Joumal of Systematic and Evolutionary Microbiology 59 (2009)7-12.
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Vancanneyt, and J. Swings - Identification of lactobacilli by pheS and rpoA gene sequence analyses, Intemational Joumal of Systematic and Evolutionary Microbiology 57 (2007) 2777-2789.
9. S. M. Naser, F L. Thompson, B. Hoste, D. Gevers, P Dawyndt, M. Vancanneyt, and J.
Swings Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151 (2005) 2141-2150.
10. N. Saitou, and M. Nei The neighbour-joining method: a new method for reconstmcting phylogenetic trees. Mol Biol Evol 4 (1987) 406-425.
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PhSn tich trinh tw gien Phes cho viec xSc djnh loSi vi khuan lactic sinh bacterioein
12. Duomg Van Hgp, Nguyin Ldn Dung Phdn loai vi sinh bdng sinh hpc phdn tir, Vietsciences, 27/02/2007; http://baigiang.violet.vn/present/showprint/entry_id/215088.
SUMMARY
PHES GENE SEQUENCE ANALYSIS FOR THE IDENTIFICATION OF A LACTIC ACID BACTERIUM PRODUCING BACTERIOCIN FROM NEM CHUA The aim of this study was to identify species level of producing bacterioein strain isolated from Ha Noi nem chua (DV4.10) by pheS gene sequencing analysis. Genomic DNA DV4.I0 was exfraeted. Then pheS gene was mn the first PCR with PheS-21-F (5'-CAYCCNGCHCGYGAYATGC-3') and PheS-22-R (5'-CCWARVCCRAARGCAAARCC- 3'). PCR product was checked by by electrophoresis on a 1% (wt/vol) agarose gel at 75 V for 45 min with marker (SmartLadder, Eurogentec. Searing, Belgium). PCR product of DV4.10 collected a bold band al 450 bp position similar lo size of pheS gene [6]. This PCR f oduet continued to mn the second PCR with seperaled primer, purified, sequenced and eomp uxted to sequencing of known sequence database in EMBL or BCCM/LMG collection i Gent university, Belgium. The result showed that DV4.10 is Lactococcus lactis subsp. laclis species.
Thus, the pheS gene sequence provides a useful tool. It have a higher discriminatory power for reliable identification of species level.
