Tiiu ban Sinh hgc vd ngudn lgi sinh vdt Biin
DA DANG QUAN XA VI KHUAN N 6 I 6 MQT SO KHU VU^C VEN BIEN PHiA BAC VIET NAM
Pham Th^ Thu', Corinne Bouvier^, Chu Van Thuoc' va Trinh Van Quang' 'vien Tdi nguyen vd Mdi Trucmg Bien, 246 Da Ndng, TP. Hdi Phong
^Trung tdm Nghien cuu Khoa hoc, qu6c gia Phap (CNRS) Email: [email protected]
Tdm tdt:
Vi khudn phdn bo rgng khdp trong cdc mdi tru&ng tu nhiin vd sinh vgt, chung ddng vai trd quan trgng trong tu nhiin vd cugc sdng cua con ngu&i.
Chung cd mdi tirang quan. tuang tdc chat che vai nhau, v&i cdc ti bdo vdt chu vd v&i cdc yiu td mdi tru&ng trong chuc ndng vd cdu trite ciia cdc hi sinh thdi. O Viit Nam nghien cihi vi qudn xd vi khudn biin cdn it. Dgc biit, chua cd nghien cuu vi sinh thdi vd da dgng qudn xa vi khudn biin bdng cdc phuang phdp sinh hgc phdn tit hiin dgi nhu PCP-DGGE. FISH... Bdi bdo
trinh bdy mgt phdn ket qud dp dung cdc phucmg phdp PCR, DGGE vdphdn mim chuyen dung (Quantitive one - Bio Rad. XLSTAT 2010) trong phdn tich da dgng qudn xd vd tuang quan v&i mdi truang ciia qudn xd vi khudn ndi & mdt sd khu vuc ven biin phia bdc Viit Nam.
DIVERSITY OF BACTERIAL COMMUNITIES IN SOME NOTHERN COASTAL AREAS OF VIETNAM Abstract:
The bacteria widely distributed in the natural environment and living organisms, they play an important role in nature and human life. They have relationships, interact closely with the host cell and with environmental factors In the function and structure of ecosystems. In Vietnam, the study of
marine microbial communities was few. In particular, no studies on diversity and ecology on marine microbial communities using molecular methods such as PCR-DGGE, FISH... This paper presents some result of application of PCR, DGGE methods and specialized software (Quantitive one Bio Rad, XLSTAT 2010) in analyzing diverse communities and environmental correlates df bacterial communities in some coastal areas north of Vietnam.
1. MO DAU
Vi khuan phan bd rdng khdp trong cac mdi trudng tu nhien va sinh vdt, chiing dong vai tro quan trpng trong tu nhien vd cupc song ciia con ngudi. Chung tham gia vd gdp phan kh^p kin cac chu chinh vdt chdt trong tu nhien, ddng vai trd quan trpng trong qud trinh tu ldm sach cua.cac he sinh thai thuy vyc, ddng gop mdt phan quan trpng trong da dang sinh hoc. Nhung vi khuan ciang la nguyen nhdn gay bdnh vd gdy chdt cho nhieu qudn
Hgi nghi Khoa hgc vd Cong ngh^ biin todn quoc ldn thir V
thd sinh vat chu, ldm dnh hudng tdi su da dang di tniydn va tidn hda cua cac qudn thd vat chii ... Chiing cd mdi tuong quan, tuong tdc chat chd voi nhau, voi cdc td bdo vat chu va voi cdc ydu td mdi truong trong chuc nang vd cau tnic cua cdc he sinh thdi. CJ Vidt Nam nghien cuu vd qudn xa vi khudn bien cdn it, va chii ydu su dung cdc phucmg phdp nghien cuu truyen thdng, chua co nghien cuu vd sinh thdi va da dang quin xa vi khuan bidn bdng cdc phuong phdp smh hoc phdn tu hien dai nhu PCR-DGGE, FISH....
Do vdy, trong dd tai cap Vien Khoa hpc vd Cdng nghe Viet Nam nghien cuu vd
"Nghiin cihi cdu triic vd biin ddng qudn xa vi rut vd vi khudn ndi trong mdi tru&ng ven biin phia bdc VietNam... ", thi ndi dung da dang vd cdu tnic quanxa vi khuan ndi da dugc ddt ra. Bdi bdo ndy trinh bdy mdt phdn kdt qua nghien cuu tai cua sdng Bach Ddng - Hai Phdng, cua sdng Ba Lat - Thdi Binh, bai bidn Sdm Son - Thanh Hda, dugc thu vao miia ' mua 7/2010, vdi 12 tram tren 3 mat cdt thu mdu theo chidu sdng-bidn.
2. PHU'OfNG PHAP NGHIEN CUtJ 2.LStfdothumau
Hinh 1. Sa dd thu mdu 2.2. Phuong phap thu vd bao quan mau
- Cdc thdng sd mdi tnrdng (nhiet dp, dp man, pH, DO) duac do bdng may CTD - Mau nuoc dugc thu bang Bathomet J »i^
+ Nude dugc chilt vao cac chai vd cd djnh hda chit vdi cdc mlu phdn tich hda hoc + Vol mau dung cho phuang phdp sinh hpc phdn tir dugc loc tnrc tiip 4 5 r a H i f . lpc polycacbonate voi lo 0,0 nni, hong khd mdng vd bdo q i L ttong dilu M d n T ^ I n T '
Cac mau dugc bao quan 1 ^ vd chuyen ngay vl phdng thi nghiim dl tiip ^^^^ ^^
Jiiu ban Sinh hgc vd nguon lgi sinh vdt Biin
2.3. Phffong phdp phan tich mau
2.3.1. Phucmg phdp tinh lirang vi khudn ting si bang phumtg phdp nhugm phan lir s l lugng vi khudn dugc nhugm vdi mdi phan tii SYBR Gold theo phuang phdp cua Patel vd cdng su [4]. Sau dd dinh Iugng tten kinh Men vi hu^fuh quang d dp phdng dai lOOOx.
Hinh 2. So do thi nghiim 2.3.2. Phircmg phdp PCR - DGGE
Ky thudt DGGE dugc sir dung ttpng nhieu nghidn cihi da d?ng vi sinh vdt vd phdt hien dot bien, chiing cd kha ndng tdch ridng cdc ADN khdc nhau vd thdnh phdn vd trinh tir nucleotit nhung cd cimg kich thudc [I; 6], mdi band dugc gpi Id mgt dgn vi phdn loai (OTU - operational taxanomic unit).
ADN tdng sd cua qudn xd vi khudn ndi dugc tdch chidt theo phuang phdp cua Bdstrom vd cdng str [5], va sdn phdm dugc kiem tta bdng didn di tren agarose 0,8%.
Do?in gen vimg V3 md hod 16S rRNA cila vi khudn dugc nhan len bang ky thuat PCR tir ADN ting sd vdi cap mdi 314f-GC - 518r [2]
DGGE thuc hipn tten may - INGENYphorU DNA Mutation Detection System (Ingeny International). Sdn phdm PCR dugc tach biet tten ban gel 8%i (wt/vol) actylamide/bis- acrylamide vdi gradient ciia chdt biin tinh ADN tir 40%i din 70% (100%> chdt biin tinh Id 7M urea vd 40% formamide) vdi ddng dien lOOV trong 18h tai 60°C trong dem Ix TAE (Euromedex). Gel DGGE duoc nhudm vdi SYBR Gold, chup dnh tten bdn soi UV (GelDoc).
2.4. Phirong phdp xir Iy sl lifu
- s l lipu va biiu dd dugc xir ly vdi phdn mim Quatity one (Biorad), Microsoft Office (Excel) vd phdn min thing kd XLSTA T2010.
• Xdy dung cay cdu tnic qudn xa ttong khu vuc nghien cihi bdng phucmg phdp phan nhdm (UPGMA) d\ra tten hS sd di truyin tucmg ddng.
Hgi nghi Khoa hgc va Cdng nghj biin loan quic ldn thic V
3. KET QUA VA THAO LU^JVf 3.1. Phan bo so luong te bao vi khuan tong sl trong mua mua (7/2010)
"ii-
Hinh 3. Biin ddng vi khudn tdng sd theo khdng gian vd thai gian Hinh 3 ben phdi cho thdy, d cd ba mat cdt nghien cull diu cd chung mpt xu hudng, qudn xa vi khudn biin ddng gidm dan theo chilu tir mdi trudng dam nudi tdm ra mdi trudng ben ngodi, va tir bd ra ngodi blln. Mat dp cua vi khuan giao dpng trong khoang 2x10'tb/ml tai 4x10* tb/ml (bdng 1). Hinh 3 ben ttdi cho thdy, MCI vd MCII cd su tuang quan chdt (R2 = 0,6), nhung d MCI vd MCIII thi khdng cd su tuong quan.
Bdng 1. Mat dp vi khudn ttong viing nghien cihi vdo miia mua Mat dd (tb/ml)
Cao nhdt Thdp nhdt Trung binh
Bach Ddng (MCI) 3.E+06 5.E+05 2.E+06
Ba Lat (MCII) 2.E+06 6.E+05 l.E-i-06
Sdm San (MCIII) 4.E+06 I.E+06 2.E+06 3.2. Da dang va cau triic di truyen ciia quan xd vi khuan noi khu vu-c nghien ciru 3.2.1. Tich chiit AND ting si tir cic miu vi khuan nil khu vtfc nghien ciiru
San phdm ADN ting sl ciia 19 mdu vi khudn nli d cdc khu vuc nghien ciiu dugc tdch chiet, va kiem tta bdng dien di tren gel agarose 0,8%., kit qua thi hien tten hinh 4.
Hinh 4. Bdn diin di kiim Ira sdn phdm ABN tdng si
Tiiu ban Sinh hgc vd nguin lgi sinh vgt Biin
Tir kdt qud tren hinh 4 ta thay, tat ca cdc mdu tdch chiet ddu co ADN nhung ham Iugng ADN khdng cao, vd cd su dut gdy ADN. Nhung dii lugng ADN cho phdn iing PCR.
3.2.2. Nhan dogn gen dich bdng ky thugt PCR
Doan gen vung V3 ma hoa 16S rRNA qua phan ling chuoi trung hgp PCR va sdn phdm dugc kidm tra trdn ban gel didn di agarose 2%, kdt qua dugc thd hien tren hinh 5.
Hinh 5. Bdn di^n di kiim tra sdn phdm PCR
TIT ket qud tren hinh 5 cho thdy, cdc bang thu dugc tuong ddi ddc hi?u, kich thudc cua cdc bang phii hgp vdi kich thuac theo tinh todn ly thuylt (200 pb), va dii chit Iugng dd tiln hdnh nghidn ciiru tiep theo.
3.2.3. Bien di bien tinh doan gen dich tren gelAcryamide
Sdn pham PCR cua cac mau dugc phan tich bing dien di biin tinh, vd kit qua dugc thd hi|n tren hinh 6.
Hinh 6. Bdn diin di biin tinh (DGGE) cita cdc mdu nghiin cihi Hinh 6 cho thdy, tten moi mSu ddu dugc phdn chia thanh nhieu band (v^ch hay don vi phdn loai - OTU: operational taxonomic unit) khdc nhau, dilu nay cho thdy ciing mdt kich thudc doan ADN idiung lai chijra nhilu trinh tu vd thdnh phin nucleotite khac nhau, ddng nghTa vdi qudn xa vi khuan trong moi mau cd su da dang nhom vi khudn, va Ioai vi khudn.
Hgi nghi Khoa hgc vd Cdng nghj biin ' " " " T'''''-'"" '^"' ^
Mdt khdc cac band l»i cd dd ddm nhat khdc nhau tten bdn dipn di, dilu ndy thd hien ham luang ADN cd teong band, hay sd Iugng cd thi vi khudn cua timg dgn vj phdn loai c" ttong miu cung cd sukhdc nhau. Ddc biet ttong cac mdu thugc MCI vd mdu Sl-M, B4 va Bl-M cd sir xuat hien OTU uu thi tai band 21. Nhin chung, khi xdt ve dg dam vd so lugn| cua cdc OTU/mdu thi xu thi vdn cd xu hudng gidm theo hudng sdng bien ttdn ttmg mdt cdt, va ting mdt cao hon tdng day, dilu nay cimg phii hgp vdi cdc nghien ciiu cua cdc tdc gia tnroc ['!•
3.2.4. Phan bi va si Itr^ng cic dan vjphttn logi cia quan xd vi khuan khu vifc nghien ciru
Bdng 2 Suphdn bd vd sa lugng cdc bdng (OTU) tren timg mdu nghiin ciru
Til kit qud bdng 2 cho thdy, sl lugng OTU cia cdc mau nghien eihi dao dpng ttong khoang 7-16 don vi/mlu, cao nhdt d ttam S3-D (16 dom vi), thip nhdt tai trani B3-D vd D2-M (7 don vi). Nhin chung, si lugng OTU t?i cdc mdt cdt giam ddn theo hudng sdng bien nhung ttong mdi trudng ddm nudi tdm va mdi trudng ngodi, ciing nhu ting mat vd tdng ddy thi lai chua thdy quy ludt rS rdng so vdi biin ddng sd lugng tl bdo tdng sd (hinh Mat khdc, tan sd b&t gdp cdc OTU ttong cdc mau nghien cim thi hien nhdm vi khudn, lodi vi khudn dd dugc phan bl rdng hay hep ttong khu vuc nghien ciiu, kit qud bdng 2 cho thdy, don vi phan lo?i 22 (band 22) bdt gap d tdt ca cdc mdu (19 mau), tiip theo la dan vi 28 (cd d 18 man), dan vi 23 (16 mau), cd 4 lo^ OTU chi bdt gdp mdt ldn... dilu ndy cho thdy sir da dang cdc nhdm lodi vd lodi cd ttong jiuan xd vi khudn nli khu vuc nghien eiiu Id rat da dang.
Tiiu ban Sinh hgc vd nguon lgi sinh vdt Biin
3.2.5. ciu trdc quSn xa vi khuan noi khu vuc nghien ciru
Bdng 3. Hi sd tuang dong di truyin giiia cdc mdu nghiin ctiu
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Tir ket qua bang 3 cho thdy, he sd tuong ddng di truyen cao giiia cdc mau chii yeu dugc thdy giiia cdc mdu df ciing mpt mat cdt, dieu nay the hien su tucmg ddng cao va cd su khac biet IdTi giQa quan xd vi khuin tai cdc mdt cdt nghien cuu, vdi cac ddc didm tu nhien vd mdi trudng khdc nhau. Tu he sd tucmg ddng di truyen, mdi lien he di truyen giiia cdc mau, mdt cdt trong quan xa vi khuan ndi khu vuc nghien cdru dugc xay dung d^ra tren phucmg phdp phdn nhdm (UPGMA), vd kdt qud dugc the hien tren hinh 7.
Hinh 7. Quan hi di truyin ciia qudn xd vi khudn khu vuc nghiin cuu dua trin dogn gen V3 md bodies rARN
Tu hinh 7 cho thiy, qudn xa vi khuin ndi khu vuc nghien ciiu dugc chia lam 5 nhdm khac nhau, vcri nhdm 2, nhdm 4 va nhom 5 ddc trung ldn lugt cho 3 mat cdt thu mau (MCIII - Sdm Scm, MCI - Bach Ddng, MCII - Ba Lat), con lai nhdm 1 thd hien ddc trung cho mdi trudmg dam nudi tdm viing ciia sdng, nhom 3'cd thd la dac trung cho mdi trucmg
Hgi nghj Khoa hgc vd Cong nghi biin todn quocJd;lj!BJL
cd su sao trdn Ddc biet, nhdm 5 cd sir ddc tnmg thudn cho MCII vd vi khuan noi tai cac team' cd su tuong ddng di tniyin tdi 80-90%, ngugc lai thi MCI cd su bien ddng trong cau tnic di ttuyin cua quin xa ldn, cho thay sir bien ddng cua dieu kien mdi tmang cua khu vuc nay cao, ddn din tuang ddng di ttuyen ciia cac qudn xa vi khudn tai cdc tram la thap.
3.3. Tuong quan ciia cau true quan xa vi khuSn nli vdi mgt so yeu to moi trudng Bdng 4. He sl tuang quan (R^) cua sd lugng tl bdo vd OTU vdi cdc ylu td mdi trudng
Yeutd OTU Vi khudn (tb/ml)
Nhiet dp'(°C)
"pH "
DO (mg/L)
"S%o N-NOj- (ng/L) N-N03"(ng/L) N-NH4* (tig/L) P-PO?-(tig/L)
Si67""(MR/L) BODs (mg/L) 1 C - hiru ca (mg/L)
Bach Dang (MCI) OTU
1 0,158"
0,262 ' 0,019 "
0,155
"o^dsi' 0,002 0,060"
0,005 6,019 0,291 0,015 0,028
Vi khuan (tb/ml)
0,158
" 1 ' 0,039
0,869 0,816 0,383 0,736 0,522 0,672 0,630 0,440 0,626 0,903
Ba Lat (MCII) OUT
1 0,112 0T628 0,676 "
0,393 0,970 0,321~1 0,274 0,080 '6,052 0,877 0,029 0,083
Vi khudn (tb/ml)
0,112 1 6,640 0,555 0,181 0,230 0,830 0,819 0,859 0,769 0,197 0,725 0,752
Sdm San (MCIII) OUT
1 0,322
"6,053 0,074 0,021 0^662' 0,054 1
"6,055^
0,024 0,023 0,004 0,001 0,006
Vl khuan (tb/ml)
0,322 1
" 0,254 0,243 0,148
"0,174
" 6,278 6,288 "
0,224
"0,219 0,134 0,146 0,072 Tir ket qua tren bdng 4 cho thdy, nhin chung sd lugng te bdo vi khudn ndi tdng sd tai MCII vd MCI cd su tuong quan vdi nhieu yeu td mdi trudng, yeu td cacbon hihi ca tdng sl, pH, DO la nhimg yeu td cd sir tuong quan chat che d MCI, cdn MCII Id cdc loai mudi dinh dudng, ngupc lai d MCIII khdng thay cd su tucmg quan. Xet vdi sd lugng don vj phan loai, su tucmg quan chat chi thdy xudt hien vdi ylu td do man. Silicate vd pH tai MCII.
Dieu ndy cho thdy, cac yeu td mdi trudng trong khu vuc nghien cim it anh hudng tdi so lupng don vi phdn loai, vd it anh hudng tdi sir phdn bd vl sd lugng tl bdo vi khudn nli tdng so trong khu vuc MCIII.
4. KET LU^N
1. Mat do cua vi khudn ndi tdng sd trong khu vuc nghien cuu dao dpng tir 2x10* tb/ml tdi 4x10 tb/ml, vd xu hudng bieii ddng chung cua mat dp vi khudn ciing nhu don vi phdn loai gidm dan theo chieu sdng biin, nhung tir mdi truong nudi thuy sdn tdi mdi trudng ciia sdng, tdng mat tdi tdng ddy thi chi thdy ro xu hudng vdi mat dp vi khudn.
2. Cd su da dang ve trinh tu vd thanh phin nucleotite tren vung gen V3 ma hod 16S rRNA ciia qudn xa vi khudn nghien curu, vdi su da hinh vl sd lugng ciia cdc OTU, dao ddng trong khodng 7-16 dan vi/mau, cao nhlt d tram S3-D (16 dan vi), thdp nhlt d tram B3.b va D2-
Tiiu ban Sinh hgc va nguon lgi sinh vgt Bien
M (7 don vi), OTU uu thd tai vi tri band 21 trong cdc mdu thudc MCI vamdu Sl-M, B4 vd Bl-M. Tdn sd bdt gdp va phdn bd OUT/mdu dao dgng trong khodng 1 - 19, trong do OTU 22 xuit hien d cd 19 mdu.
3. Qudn xd vi khudn ndi khu vuc nghien cuu dugc chia ldm 5 nhdm khdc nhau, vdi nhdm 2; 4; 5 d^c trung cho 3 mat cdt thu mau (MCIII, MCI, MCII), cdn lai nhdm 1 dac tnmg cho mdi trudng nudi thiiy sdn vung cua sdng, nhdm 3 cho mdi trudng co su xdo trdn.
4. OUT chi cd su tucmg quan chdt vdi yeu td dp man, Silicate vd pH tai MCII, cdn mat do vi khudn ndi cd su tuong quan chdt chd voi ydu td cacbon huu co tdng sd, pH, DO a MCI, MCII la cdc mudi dinh duomg.
TAI LIEU THAM KHAO
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2. Muyzer, G., Waal, E.C.D. and Uitterlinden, AG., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695-700.
3. Ovreas L., Forney L., Daae FL. and Torsvik V, 1997. Distribution of bacterioplankton in meromictic Lake Saeienvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl.
Environ. Microbiol., Vol 63, No. 9. 3367-3373.
4. Patel A, Noble RT, Steele JA, Schwalbach MS, Hewson I, Fuhrman JA., 2007. Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I. Nature Protocols 2:269-276.
5. Kjarstin H. Boshom, Karin Simu, Ake Hagstrdm, and Lasse Riemann, 2004.
Optimization of DNA extraction for quantitative marine bacterioplankton community analysis. Limnol. Oceanogr.: Methods 2, 365-373.
6. Yosida N., Takahashi N., Hiraishi A., 2005. Phylogenetic characterization of a polychlorinated-dioxm-dechlorinating microbial by use of microcosm studies. Appl Environ Micobiol. Vo. 71. 4325-4334.
