PETROVIETNAM Phan tfch cac diem dot bien tren gen 16S rDNA cua vi khuan phan lap tii mo Bach Ho sau khi xCrlybdiHexatreatlSU TS Dam Sao Mai Vien Cdng nghe Sinh hgc vd ThUc pham, Dgi hgc Cdng nghiep TP[.]
PETROVIETNAM Phan tfch cac diem dot bien tren gen 16S rDNA cua vi khuan phan lap tii mo Bach Ho sau xCrlybdiHexatreatlSU TS Dam Sao Mai Vien Cdng nghe Sinh hgc vd ThUc pham, Dgi hgc Cdng nghiep TP.HC GS.TSKH.Vu Dinh Huy Trudng Dgi hgc Bdch Khoa, Dgi hgc Qudc gia TP.HCM TSKH Abdurakhmonov I.Y., Vien si Abdukarlmov A Vien Di truyen hgc vd Sinh hgc thUc vdt thUc nghiem, Vien Hdn Idm khoa hoc Uzbekistan Tom tat DUa tren cdc phdn tich ve di truyen, chung tdi dd xdc dinh dffpc chung vi khudn phdn lap tff mo Bach Ho sff dung nghien cffu ehinh la Klebsiella pneumonia DUdi tdc dpng cua ehdt diet khudn (Hexatreat 1512), cdc chung hoang dai dd ed mpt vdi bien doi ve di truyen Mpt vdi diem SNP (single-nucleotide polymorphism) vd dpt bien diem dang indel (insertion/deletion) tren trinh tU 16S rDNA giffa cdc chdng Klebsiella sp hoang dpi vd chung dd xffly vdi chd't diet khudn dd dffdc tim thay Ket qud ndy cho thdy, cdng nghiep ddu khi, chd't diet khudn sff dung cd tdc dpng nhdt dinh den nucleotide cua doan gen 16S rDNA cdc vi khudn khdo sdt DAU KHi - s o 3/2011 HOA - CH^ BifN D A U KHJ I.Mddau Mpt nhUng van de ldn ma nen cdng nghiep dau phai ddi mat la hop chat lUu huynh cd dau thd Cae hpp chat lUu huynh khdng ehi lam giam gia trj thuong pham cua dau ma cdn gay an mdn cae thiet bi khai thac dau va lam d nhiem mdi trudng Hpp chat lUu huynh dau thd ehu yeu dupe sinh qua trinh hoat ddng sdng cua vi khuan Nhieu nghien cUu da dupe thUe hien chuyen sau ve hien trang an mdn vi khuan gay nen tren he thdng khai thac, van chuyen; he thdng bdn ehUa cdng nghiep san xuat dau md tren the gidi va tai Viet Nam [1,2,3] Oe tim hieu sau ve qua trinh nay, cac nha khoa hpe da tien hanh rat nhieu nghien eUu ve he vi khuan sinh cac hpp chat ehUa lUu huynh [4] Tuy nhien, cae nghien eUu mdi ehu yeu dUng lai d tae hai cua vi khuan gay an mdn, ehU chUa di sau vao ban chat cua chinh vi sinh vat qua trinh an mdn Vdi sU phat trien cua khoa hpe hien dai, qua trinh phan tich di truyen da gdp phan vao viec xae dinh chinh xae hon eae vi khuan cung nhU nhUng thay ddi nha't djnh ve di truyen cua ehung Muc dich nghien cUu eua chung tdi la: -I- Xae djnh mdt nhUng chung vi khuan ed kha nang san sinh hpp chat luu huynh md dau Bach Hd; -I- Khao sat tae ddng cua chat diet khuan thuong mai Hexatreat 1512 len sU thay ddi gen 16S rDNA eua chung vl khuan Ddi t U P n g nghien edu la hai ehung vi khuan gay an mdn (5KK va 8KK) phan lap dupe td cac mau dau lay tU md Bach Ho; sd dung mdi trUdng postgate B de phan lap cae vi khuan cd mau dau; tach cae chung tao khuan lac ed mau den, bieu hien sU tao hop chat lUu huynh - lam giau eae chung mdl trudng postgate A Sd ' dung mdt lUpng vi khuan cd kha nang sinh hop chat luu huynh nhU nhau, nudi cay cung mdt che dp, dUa tren lupng hpp chat lUu huynh hinh va cac phan Ung thd nghiem dp an mdn de xae djnh chung ed kha nang an mdn eao nha't cho cac nghien eUu ke tiep Cac thUc nghiem dupe thUe hien ea d dieu kien ky va dieu kien hieu Cac phUPng phap thUe nghiem da dagc edng bd tren mdt sd tap ehi [5,6] II Phuong phap nghien cuTu 2.2 Chuan bi moi tri/oing nuoi cay 2.1 Vat lieu: dau thd lay td md Bach Ho nam tren them luc dja mien Nam Viet Nam Cac mau dau ed ham lupng paraffin la 30%, cd chUa vet cua lUu huynh, 20% metan va rat nhieu ion sulfate (16,46 - 617,25 mg/L), theo vj tri lay mau 52 DAU KHi -SO 3/2011 Nudi cay cae ehung 5KK va 8KK mdi trUdng postgate A [7], gdm: kali dihidrophosphat (KH2PO4) 0,5g, amon elorua (NH4CI) Ig, natri sulfur (NajS) 0,1 g; eanxl sulphat (CaS04.2H20) PETROVIETNAM Ig; manhe sulphat (MgS04.7H20) 2g; natri laetat 3,5g; djeh ehle't nam men I g ; sat sulphat (FeS04.7H20) 0,5g, natri elorua (NaCl) 5g, them nUde eat den 1000ml, dieu chinh dung djeh den pH = 7,2-7,4.TUdd, thu dupe dung djeh vi khuan 5KK va 8KK Cho chat diet khuan thuong mai "Hexatreat 1512", ndng dp 60ppm (theo the tich) vao dung dich vi khuan 5KK va 8KK cd mat dp 10^ vi khuan/ml Sau 48 gid tae ddng bdi chat diet khuan, chung tdi da dung phuong phap quang tai bude sdng 580nm [8], khdng phat hien thay vi khuan nao cdn sdng sdt NhUng bang phuong phap dia tren mdi trudng postgate B [7], ehung tdi phat hien thay edn mdt vai vi khuan hoang dai sdng sdt, ehUng td ehung da thich nghi vdi mdi trudng cd mat chat diet khuan Nhu'ng vi khuan da thich nghi vdi chat diet khuan 6\Jgc viet tat la 5KK(B) va 8KK(B) Lam giau cac ehung 5KK(B) va 8KK(B) tren mdi trudng postgate A, thu dugc dung dich vi khuan 5KK(B) va 8KK(B) 2.3 Dinh danh va so sanh trinh tUgen 16S rDNA cua cac vi khuan Tach doan gen 16S rDNA cua vi khuan lUa chpn, tinh sach DNA, khuech dai doan gen 165 rDNA cap primer 27F (5'-AG AGTTTG AT CM TG GC TCAG-3') va 1525R (5'-AA GG AG GT GW TC CA RCC-3') [8][9] M=C:A, R=A:G, W=A:T; all 1:1 Quy trinh PCR dUpc chay theo chu trinh sau: 94''C 90s ^ \54.5''c/ 72''C 72''C 90s 60s 35 cycles • Nhan ban (clone) va phan tich trinh tU doan 1,5kb eua 16S rDNA cua vi khuan khao sat bang cap primer 27F (5'-AGAGTTTGATCMTGGCTCAG3'), 1525R (5'-AA GGAGGTGWTCCARCC-3'), 520F (5'-CAGCAGCCGCGGTAATAC-3'), 920R (5'-CCGTCAATTCATTTGAGTTT-3'), M13F, M13R Chu trinh PCR nhu sau: H6A - CH^ BI^N DAU KHi 96''C > 95°C 60°C 30 s ^, \ „ / \ 55"C / 15s < 35 cycles ^ Sd dung phan mem SEQUENCHER 4.2 (Gene Codes, USA) de phan tich mau, sau dd dung phan mem BLASTN de so sanh vdi ngan hang Gen (GeneBank) tim vi khuan phu hpp SCr dung CLUSTALXva Neighbor Joining algorithm vdi Kimura-2 parameters cua phan mem PAUP version 4.0b10 danh cho Macintosh de xae djnh cay phat sinh loai [9] III Ket qua va thao luan 3.1 Phan ti'ch trinh tU gen 16S rDNA cua cac mau vi khuan Ml M2 SKK iKXm C m 2000 bp 1500 b p — > 1000 bp ^tm 8KK S1£1C(B) C I mmm Oe xae lap sU khae biet ve 16S rDNA cua cac vi khuan lua chpn, san pham PCR da dupe phan tach bdi agarose 1% Ket qua eho thay, cac vach hien thj cung vj tri, chdng td cae ehung vi khuan khao sat la eung loai va eho ket qua dien di tai 1,5 kb (amplicon) [Hlnhl ] Cac doan gen DNA tU san pham PCR ndi tren dupe lam tinh sach bdi PEG juice de loai bd mdi (primer) Sd dung pCR4-TOPO-TAeloning vector de nhan ban cac DNA Cac CFU (cloning forming unit) cua DNA hinh tren mdi trUdng kanamycin dupc lam giau tren mdi trUdng LB (cd chUa 50mg/L kanamycin) Cac plasmid DNAs hinh se dupe gan vdi EcoRI 28 CFU cua vi khuan 5KK, 5KK(B), 8KK va 8KK(B) 6isgc lUa chpn de tiep tue khuech dai va phan tieh tnnh tu Ky thuat Big Dey vdi mdi (primer) Ml dUpesLrdung cho ca hai chieu xudi va ngUpc Cae san pham tao dUcic phan tach bdi agarose 1% Cae mau sau kiem chdng bdi qua trinh cloning eae DNA va xd ly cac plasmid nham dam bao dp tinh sach cua miu kiem, se dupe dem di chay trinh tU DNA bang thiet bj 3130 x Genetic Analyzer (A&B Applied Biosystem) 3.2 So sdnh trinh tff gen 16S rDNA cua cdc vi khudn chpn Ipc Khi so sanh cac trinh tU 16S rDNA da dUpc ket hpp va sda ldi cua cae vi khuan 5KK, 5KK{B), 8KK va 8KK(B) bang phan mem SEQUENCHER 4.2, chung tdi tlm vai SOObp ô>ôã diem SNP va indel point mutation tren trinh tU 16S rDNA giOa cae chung hoang dai va ehung da xd ly vdi chat diet khuan (Hinh 2, Bang 1) Ket qua cho thay 250 bp — • chat diet khuan ed tac ddng nhat djnh den nucleotide eua doan gen 16S rDNA cac vi khuan khao sat Hinh Sdn pham PCR tren agarose % gen I6S rDNA (Hinh va Bang 1) cua cdc vi khudn lUa chgn Mhlkb DNA ladder; M2: Lambda Hindlli digested DNA ladder, C: Mdu kiem chdng 750 bp —-^ 54 DAUKHi - s o 3/2011 PETROVIETNAM Bang So sdnh sukhdc biet ve nucleotide cua cdc vi khuan VI trf cua cac nucleotide 5KK 5KK(B) 10 A T T C A G 522 A G 523 G C 532 C T 533 G C 778 A 859 T 8KK(B) 5KK 8KK T C C T C T T C T A A T intion delition G A C C T C A G A G 281 406 521 671 n08 1134 1359 1503 A 5KKva8KK 8KK 141 167 8KKva8KK(B) 5KKva5KK(B) G A C T G A G 3.3 So sanh sU khae biet giiira cac vi khuan chon loc bang cay phan loai De djnh danh ehinh xae eae vi khuan khao sat, chung tdi so sanh cae nucleotide cua doan gen 16S rDNA 6ugc giai ma nhd ngan hang sd lieu gen (GeneBank database) Cac chung 5KK, 5KK(B), 8KK va 8KK(B) ed trinh tU den 99% gidng vdi Klebsiella spp., dd gan nha't phai ke den Klebsiella pneumoniae subsp pneumoniae MGH 78578 DUa tren trinh tU 16S rDNA cua eae vi khuan khao sat, vdi UPGMA va NeighborJoining algorithms eua phan mem PAUP, da ehi rang eae chung Klebsiella sp nam chung nhdm Mac dau vay, ehung van cd nhdng diem khae biet nha't djnh (Hinh 4) Cae vi khuan 5KK, 8KK va 5KK(B), 8KK(B) dugc xae lap tren cay phan loai the hien d Hinh DUa vao ket qua phan tich, nhan thay: - Du eho cd vai diem SNP tren trinh tU 16S rDNA giUa hai chung 5KK va 8KK, nhUng khdng ed sU sai khae qua nhieu tren cay phan loai - Chat diet khuan Hexatreat 1512 ehi tae ddng len sd diem nucleotide cua 5KK va khdng tao sU sai biet ldn den chung 8KK, the hien tren cay phan loai cua 8KK va Hinh Trinh tu 16S rDNA cua 5KK, 5KK(B), SKKand 8KK(B) (phdn mem ClustaiX) 8KK(B) Tuy nhien, sai biet DflU ¥M\ - s o / 1 HOA - CH^ Bl^N D A U KHI (cytoplasm), cac chat eung co kha nang tac ddng den chat diet khuan sd dung nghien cufu, trUde tac ddng len the nhan ed chufa DNA, lam thay ddi sU tac ddng len DNA eua hai vi khuan SU tac dpng eung the hien tren lUpng vi khuan bj tied diet dudi tae ddng cua chat diet khuan; va sU thich nghi cua vl khuan nghien edu vdi chat diet khuan sd dung theo thdi gian Klebsiella pneumoniae strai Klebsiella sp P2 Klebsiella sp.PN^ Klebsiella sp KGA Klebsiella pneurronlae 5KK(B) biocide treated Nhu vay, tU nhien, sU bien dj di truyen gida cac loai la rat da dang Viec kiem soat sU bien dj cua cac vi khuan va tac ddng cua ehung den mdi trUdng can dupc theo ddi thudng xuyen de cd cac bien phap xd ly phCi hpp Klebsiella pneunnionlae 'I KI ebsi el I a pneun-oni ae strai n H R16 Klebsiella pneumeniae strain HR9 Uncultured bacterium clone SJTU,jD_02 ^0S Uncultured bacterium clone SJTU„D„03_78 Klebsiella pneunfioniaestrain2 IV Ket luan litutionsy^HIHKH^HHi Hinh Cay phdn lodi (KImura 2-parameter) di/a tren ket qud phdn tich DNA cua vl khudn J Chung tdi da phat hien thay chung vi khuan Klebsiella pneumoniae gay an mdn kim loai cd dau thd cua md Bach Ho; da cd tac ddng len kha nang thich Ung vdi Hexatreat 1512 cua vi khuan -Hexatreat 1512 tac ddng kha rd ret den chung 5KK Su phan biet 5KK va 5KK(B) dupe the hien kha rd tren cay phan loai va sUtUOng tac vdi chat diet khuan Cd the thay rang, du eho 5KK va BKK khdng khae biet qua ldn, nhUng tae ddng eua Hexatreat 1512 tren tUng chung eung khae SU khae the hien tren kha nang thich Ung vdi chat diet khuan cua vi khuan khao sat va sU khae biet ve mat di truyen eua cac vi khuan da thich nghi SU khae biet di truyen khdng nhieu, nhien tac ddng cua Hexatreat 1512 len tUng doan 16S rDNA eua tdng chung vi khuan khao sat cd sU khae biet rd net Dieu the hien mUc dp ben cua 16S rDNA cua moi ehung ddi vdi tac ddng nhan tao cung nhu tU nhien Ngoai ra, moi ehung vi khuan lai ed tinh chat khae cua mang te bao va chUa mdt sd chat khae te bao chat 56 DAU KHi - s o 3/2011 Dudi tac ddng cua chat diet khuan Hexatreat 1512, cac ehung hoang dai va ehung da thich nghi deu ed sU bien dj di truyen va cd anh hUdng nha't djnh den mdi trudng; Nghien cUu sau hon ve sU bien dj di truyen cua vi khuan den kha nang sinh hpp chat lUu huynh gay an mdn kim loai se dUoc de cap tdi sau Ldi cam on Cae tae gia tran trpng cam on Lanh dao XNLD Vietsovpetro va Vien NIPI da eho phep chung tdi ddOc lay eae mau dau thd, vi khuan, chat diet khuan de thf nghiem Tai lieu tham khao H.W.Rossmoore, 1981 Antimicrobial agents for water - based metalworking Fluids Journal of Occupational Medicine, April, Vol.23, No.4, p.247 PETROVIETNAM M.H.Czechowski and H.W.Rossmoore, 1980 Factors affecting Desulfovlbrlo desulfurieans Lactate dehydrogenase American institute biological sciences, Washington, DC, Vol.21, p.349 Vietnam Economy, Vietnam News Major oil field to flow this year - 2003/07/23 Ly Kim Bang, 1990 Research the microorganism for exploration, production and use of mineral oil and oil product Government project Code: 22A - 04 - 05, Vietnam Academy, Ha Noi, p.47 M.S.Dam, I.Y A b d u r a k h m o n o v , Z.T Buriev, A.Abdukarimov, 2008 The molecular - genetic taxanomleal analysis of the wild sulfate - reducing bacteria and the biocide acclimated sulfate-reducing bacteria strains ISSN 0042 -1685 Uzbek Biological Journal N 1, p.65 - 67 Dam Sao M a i , I.Y A b d u r a k h m o n o v , A Abdukarlmov, 2008 Compare the characteristics between the wild sulfate reducing bacteria and the biocide adaptive sulfate reducing bacteria ISSN 1019 - 8954 Uzbek Academy Report N1,p.63-65 7.Postgate J.R " ^ 1984 Edition University Press, Cambridge, p.31 Cambridge Lane D.J., Pace B., Olson G.J., Stahl D.A., Sogin M.L, Pace N.R., 1985 Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses Proceedings of the National Academy for Science 82, 6955 - 6959 Amann R.I., Binder B.J., Olson R.J., Chisholm S.W., Devereux R., Stahl D.A., 1990 Combination of16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations Applied Environmental Microbiology 56, 1919-1925 10 Angostini R.A., Young R.D, 1990 A ease history: investigations of microbial Influenced corrosion in a west Texas water flood Proceedings of the NACE Annual Conference Corrosion 90 NACE International, Houston, Tex United States, 119 11 Batista J F., Pereira R F., Lopes J M., Carvalho M F., Feio M J and Reis M A., 2000 In situ corrosion control in industrial water systems Biodegradation 11,441-448 12 Xiang Y Zhu, John Lubeek, John J Kilbane II, 2003 Characterization of Microbial Communities in Gas Industry Pipelines Applied and environmental microbiology 69 (9), 5354-5363 13 Nwekel CO., Okpokwasili G.C, 2003 Drilling fluid base oil biodegradation potential of a soli Staphylococcus species African Journal of Biotechnology (9), 293-295 DAU KHi - S C ... 8KK(B) 2.3 Dinh danh va so sanh trinh tUgen 16S rDNA cua cac vi khuan Tach doan gen 16S rDNA cua vi khuan lUa chpn, tinh sach DNA, khuech dai doan gen 165 rDNA cap primer 27F (5''-AG AGTTTG AT CM... Applied Biosystem) 3.2 So sdnh trinh tff gen 16S rDNA cua cdc vi khudn chpn Ipc Khi so sanh cac trinh tU 16S rDNA da dUpc ket hpp va sda ldi cua cae vi khuan 5KK, 5KK{B), 8KK va 8KK(B) bang phan... Phan ti''ch trinh tU gen 16S rDNA cua cac mau vi khuan Ml M2 SKK iKXm C m 2000 bp 1500 b p — > 1000 bp ^tm 8KK S1£1C(B) C I mmm Oe xae lap sU khae biet ve 16S rDNA cua cac vi khuan lua chpn, san