Đánh giá đa dạng sinh học vi sinh vật liên kết với hải miên tại vùng biển đà nẵng việt nam thông qua nghiên cứu metagennomics bằng kỹ thuật 16s rRNA miseq sequencing

I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - NGUYN MNH HNG NH GI A DNG SINH HC VI SINH VT LIấN KT VI HI MIấN TI VNG BIN NNG VIT NAM THễNG QUA NGHIấN CU METAGENOMICS BNG K THUT 16S rRNA MISEQ SEQUENCING LUN VN THC S KHOA HC H Ni Nm 2016 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - NGUYN MNH HNG NH GI A DNG SINH HC VI SINH VT LIấN KT VI HI MIấN TI VNG BIN NNG VIT NAM THễNG QUA NGHIấN CU METAGENOMICS BNG K THUT 16S rRNA MISEQ SEQUENCING Chuyờn ngnh: Vi sinh vt hc Mó s: 60420107 LUN VN THC S KHOA HC NGI HNG DN KHOA HC: PGS.TS NGUYN TH KIM CC TS PHM C NGC H Ni Nm 2016 Li cam oan Tụi xin cam oan kt qu th hin bn lun ny l cụng trỡnh nghiờn cu thc t Ton b s liu, kt qu nghiờn cu lun l hon ton trung thc, khỏch quan, cha c cụng b cụng khai bi mt khỏc Li cm n ti ny l kt qu ca mt quỏ trỡnh hc v nghiờn cu khoa hc ca bn thõn, cựng vi s giỳp sc ca rt nhiu cỏ nhõn v th cú c thnh cụng ny, tụi xin by t lũng kớnh trng v bit n sõu sc n PGS.TS Nguyn Th Kim Cỳc, ngi ó giỳp tụi rt nhiu bng chớnh s tõm huyt v am mờ vi lnh vc nghiờn cu khoa hc Tụi cng xin c t lũng cm n chõn thnh ti cỏc cỏn b thuc phũng Cụng ngh sinh hc, vin Hoỏ sinh bin; Vin nghiờn cu khoa hc Min Trung, Vin Hn lõm khoa hc Vit Nam ó cú nhng h tr, úng gúp, t rt nhit tỡnh giỳp tụi hon thin ti Tụi tht s rt bit n ngi thõn, bn bố v ng nghip ó ginh tỡnh cm, t nim tin, h tr, ng viờn; ng thi cng l ngun ng lc to ln gúp phn giỳp tụi hon thin ti ny Vi s h tr v kin thc; cỏc ngun d liu b sung; cỏc cụng trỡnh nghiờn cu khoa hc cú liờn quan nc v trờn th gii-Tp th cỏc Thy cụ thuc khoa sinh hc; B mụn Vi sinh vt hc v c bit TS Phm c Ngc ó giỳp tụi lm nờn s thnh cụng ca ti ny Tụi xin chõn thnh cm n! H Ni, ngy 27 thỏng 12 nm 2016 Nguyn Mnh Hựng MC LC M U CHNG 1: TNG QUAN 1.1 S lc v hi miờn 1.2 Vai trũ v tm quan trng ca vi sinh vt liờn kt vi hi miờn 1.3 a dng sinh hc vi sinh vt liờn kt vi hi miờn 11 1.4 S lc v gen 16S rRNA 16 1.5 S lc v MiSeq Sequencing 17 CHNG 2: VT LIU V PHNG PHP NGHIấN CU 21 2.1 VT LIU 21 2.2 PHNG PHP NGHIấN CU 21 2.2.1 Tỏch DNA 21 2.2.2 Thit k th vin 16S rRNA cho MiSeq 21 2.2.3 Phõn tớch th vin 16S rRNA 22 CHNG 3: KT QU V THO LUN 23 3.1 KT QU 23 3.1.1 Tỏch DNA metagenome ca vi sinh vt liờn kt hi miờn 23 3.1.2 Khuch i vựng siờu bin V4 ca gen 16S rRNA v gn barcodes 24 3.1.3 a dng cỏc cng ng vi sinh vt liờn kt hi miờn bin Nng 26 3.2 THO LUN 50 KT LUN V KIN NGH 56 TI LIU THAM KHO 58 DANH MC BNG V HèNH Hỡnh 1.1: Hỡnh thỏi Porifera, (Ruppert et al., 2004) Hỡnh 1.2: Cu trỳc c th hi miờn v cỏc t bo chc nng Hỡnh 1.3: Sinh sn hu tớnh hi miờn Hỡnh 1.4 a dng vi khun liờn kt vi hi miờn (Tabares 2011) Hỡnh 1.5: Mụ mesohyl ca (A) Ancorina alata, hi miờn cú vi khun liờn kt phong phỳ Hỡnh 1.6: Gene 16S rRNA cú vựng bin i nm xen gia cỏc vựng bo th Hỡnh 3.1: in di DNA metagenome ca vi sinh vt liờn kt hi miờn bin Nng trờn agarose gel 0,8% Lane 1: DN ; lane 2: DN10; lane Hỡnh 3.2: nh in di PCR khuch i vựng V4 gen 16S rRNA ca vi sinh vt liờn kt hi miờn bin Nng M: Marker DNA kb (GeneRuler); 1, 2, 3: Sn phm PCR vựng V4 ca mu DN9, DN10 v DN12, tng ng Hỡnh 3.3: Sn phm PCR sau gn barcodes M: Marker DNA kb (GeneRuler); 1, 2, 3: Mu DN9, DN10 v DN12, tng ng Hỡnh 3.4: th biu hin t l vi khun v c khun liờn kt vi ba mu hi miờn Hỡnh: 3.5: th biu hin thnh phn cỏc ngnh vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.6: th biu hin thnh phn cỏc lp vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.7: th biu hin thnh phn cỏc b vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.8: th biu hin thnh phn cỏc h vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.9: th biu hin thnh phn cỏc chi vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Bng 1: Phn mm, cỏc cụng c da trờn Web v cỏc c s d liu s dng phõn tớch metagenome Bng 3.1: Kt qu kim tra hm lng v tinh sch ca DNA metagenome Bng 3.2: Kt qu nhn dng cỏc mu hi miờn Bng 3.3 Túm tt kt qu phõn loi vi sinh vt liờn kt vi hi miờn Bng 3.4: Kt qu phõn loi vi sinh vt liờn kt vi hi miờn mc gii Bng 3.5: Kt qu phõn loi cỏc ngnh vi sinh vt liờn kt vi hi miờn Bng 3.6 Kt qu phõn loi cỏc lp vi sinh vt liờn kt vi hi miờn Bng 3.7 Kt qu phõn loi cỏc b vi sinh vt liờn kt vi hi miờn Bng 3.8 Kt qu phõn loi cỏc b vi sinh vt liờn kt vi hi miờn Bng 3.9: Kt qu phõn loi cỏc chi vi sinh vt liờn kt vi hi miờn M U Hi miờn l vt ch ca nhiu vi sinh vt v vai trũ ca nhng vi sinh vt ny thay i theo ngun dinh dng v s cng sinh vi hi miờn Da trờn nhng nghiờn cu cng ng vi sinh vt bng cỏc phng phỏp nh in di gel gradient bin tớnh (Denaturing Gradient Gel Electrophoresis_DGGE), gii trỡnh t gen 16S rRNA v lai hunh quang in-situ (Fluorescense In Situ Hybridization_FISH), ngi ta nhn thy ngoi cỏc thnh viờn ca Archaea cũn cú ti hn 25 ngnh vi khun liờn kt vi hi miờn Trong s ú cú Proteobacteria, Nitrospira, Cyanobacteria, Bacteriodetes, Actinobacteria, Chloroflexi, Planctomycetes, Acidobacteria, Poribacteria v Verrucomicrobia Cha rừ v virus hi miờn, mc dự cỏc ht ging nh virus c phỏt hin nhõn t bo ca Aplysina (Verongia) cavernicola Nhng nghiờn cu v vi sinh vt liờn kt vi hi miờn cho thy vi sinh vt cú th chim n 40% - 50% th tớch hi miờn v cng ng ny c trng cho tng hi miờn [40] Mi liờn h gia vi sinh vt vi hi miờn gn õy ang c quan tõm nghiờn cu do: (i) phỏt hin nhiu hi miờn cú liờn kt vi cng ng vi khun dy c v a dng, (ii) chỳng l mt ngun giu cỏc cht chuyn húa hot tớnh sinh hc cú ý ngha lnh vc dc phm v cụng ngh sinh hc [50] Vic phõn lp, nuụi cy vi sinh vt sinh trng iu kin c bit thng khú khn, c bit vi sinh vt liờn kt vi cỏc dng sng khỏc nh hi miờn bi mi tng tỏc gia chỳng khỏ phc Cng khụng th khai thỏc hi miờn tỏch chit cỏc hot cht vỡ ngun nguyờn liu ny cú hn, khú phc hi v gõy hy hoi mụi trng ti ny c thc hin vi mc ớch nghiờn cu s a dng ca vi sinh vt liờn kt vi hi miờn bin trờn c s phõn tớch d liu metagenome ca vi sinh vt Kt qu nghiờn cu s cung cp ngun c s d liu cú giỏ tr cho cỏc nghiờn cu tip theo v mi liờn h gia vi sinh vt-hi miờn bin CHNG 1: TNG QUAN 1.1 S lc v hi miờn Hi miờn (bt bin) l ng vt n lc, a bo, c xa thuc ngnh ng vt thõn l (Porifera) Ngnh Porifera l mt nhúm a ngnh bao gm ba lp chớnh, c c trng bi c cu gai ca chỳng: (1) Hexactinellida (glass sponges -bt bin thy tinh) cha gai silic cu trỳc hexactine; (2) Calcarea (calcareous sponges -bt bin vụi) cha gai vụi (calcareous spicules); (3) Demospongiae (demosponges) cú khong 85% cỏc loi c cụng nhn hin nay, thng cú mt b khung khoỏng cht bao gm gai silic (siliceous spicules) Tuy nhiờn, mt s loi Demospongiae khụng cha b khung [21] Hi miờn cú cu trỳc c th n gin nht tt c cỏc loi ng vt a bo: thiu c quan v mụ thc; khụng cú c, h thn kinh, h tiờu húa, h tun hon, c quan bờn trong, v kh nng ng Cỏc t bo ca hi miờn sp xp cú th t gm nhiu lp t bo chuyờn bit (Hỡnh 1.2) phự hp vi li sng n lc Hu ht hi miờn cú b khung cng c hỡnh thnh t canxi cacbonat v cỏc gai silic dioxide Cỏc si Collagen v spongin cú tỏc dng h tr thờm s cng chc v n hi cho b khung [21] Ngoi cỏc loi t bo xp khỏc nhau, mt cao ca cỏc vi sinh vt cng c tỡm thy cỏc mesohyl ca mt s loi hi miờn sõu ph bin tỡm thy hi miờn t 5-10m ng vt thõn l, ú cú hi miờn thng n bng cỏch hỳt nc qua cỏc l chõn lụng Mt dũng chy c trỡ liờn tc qua c th ly thc n, ụxy cng nh loi b cht thi Khi nc ó c lc qua cỏc khoang choanocyte, nú b thi c th qua cỏc osculum, v c bn l vụ trựng [43, 44] Hu ht hi miờn cú th bm mt lng nc tng ng vi lng c th ca nú mi giõy [47], iu ny cú th bng vi ngn lớt mi ngy [55] Mi liờn h gia vi sinh vt vi hi miờn gn õy ang c quan tõm nghiờn cu do: (i) phỏt hin nhiu hi miờn cú liờn kt vi cng ng vi khun dy c v a dng, (ii) chỳng l mt ngun giu cỏc cht chuyn húa hot tớnh sinh hc cú ý ngha lnh vc dc phm v cụng ngh sinh hc [50] *) Cu trỳc chung ca mt c th hi miờn: Porifera cú dng hỡnh thỏi c th t n gin (asconoid v syconoid) n phc (leuconoid) c hỡnh thnh bng cỏch thay i mc gp ca mng c th bờn v s thay i phc ca h thng kờnh [9] Hỡnh 1.1: Hỡnh thỏi Porifera, (Ruppert et al., 2004) Pinacoderm: B mt bờn ngoi, c hỡnh thnh bi cỏc t bo biu mụ c gi l pinacocytes lp ngoi ny nc s c rỳt qua cỏc l (Ostia) Mesohyl:Mt matrix ngoi bo gia pinacoderm v choanoderm cú vai trũ nh mt h khung dn, chim phn ln c th hi miờn Cỏc ht thc n c chuyn t choanocytes ti Hỡnh 1.2: Cu trỳc c th hi miờn v cỏc t bo chc nng archaeocytes di ng (hoc amoebocytes) thc bo Amoebocyte l nhúm cỏc t bo ton nng (totipotent), thc hin vic phõn phi dinh dng t Choanocytes ti cỏc t bo khỏc; giỳp cỏc t bo trng (Oocyte) phỏt trin, chuyn tinh trựng t choanocytes ti cỏc t bo trng; cú kh nng bit húa thnh cỏc loi t bo cú chc nng khỏc Vớ d nh collencytes, sn xut cỏc si collagen, sclerocytes hỡnh thnh spicule v spongocytes sn xut spongin Nh vy, vi sinh vt liờn kt hi miờn l c hiu cho vt ch chỳng u c ly mu t cựng mt vi trớ a lý Hai loi hi miờn Suberites diversicolor v Cinachyrella australiensis ca Indonesia c phõn tớch a dng vi sinh vt liờn kt bng pyrosequencing ó nhn dng c 29 phyla gm Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Chloroflexi, Cyanobacteria, Acidobacteria, Gemmatimonadetes, Spirochaetes, Nitrospirae, ZB3, Verrucomicrobia , Chlamydiae v Poribacteria Trong hi miờn C australiensis, OTUs phong phỳ nht thuc h Methylococcaceae Ngoi cỏc h Hyphomicrobiaceae v Rhodobacteraceae Acidimicrobidae, Gammaproteobacteria v Deltaproteobacteria nhiu hn C australiensis so vi S diversicolor Alphaproteobacteria phong phỳ hn S diversicolor so vi C australiensis [Cleary et al., 2013] Kt qu ỏnh giỏ s a dng vi sinh vt liờn kt hi miờn Cinachyrella (DN10) ó phỏt hin c 12 ngnh vi khun S ngnh ó c xỏc nh nghiờn cu ny thp hn so vi mt s nghiờn cu trc õy: 23 ngnh t SG2 Cinachyrella v 14 ngnh t SG1 Cinachyrella (Marie et al, 2014), 26 ngnh t hi miờn thuc vựng triu i Tõy Dng (Anoop v Agostinho, nm 2015) Mt s ngnh chớnh c xỏc nh nghiờn cu ny, chng hn nh Proteobacteria, Cyanobacteria, Actinobacteria, Firmicutes, Chloroflexi, Caldithrix, Nitrospirae, cng c phỏt hin t cỏc loi hi miờn khỏc (Marie et al, 2014; Anoop v Agostinho, 2015; Detmer et al., 2009 ; Jasmin et al, 2015) Nhng nghiờn cu v a dng vi sinh vt liờn kt hi miờn chi Rhabdastrella v Dactylospongia rt ớt Mt s tỏc gi nghiờn cu vi sinh vt liờn kt vi R.globostellata bng phng phỏp phõn lp v thy rng cỏc chng phõn lp phn ln thuc Alphaproteobacteria Ngoi ó phỏt hin cỏc vi khun Gram (+) cú % GC thp (ngnh Fimicutes), vi khun Gram (+) cú % GC cao (ngnh Actinobacteria) v Cytophaga-Flavobacterium-Bacteroides (ngnh Bacteroidetes) [Lafi et al, 2005] Schmitt v cs (2012) s dng gii trỡnh t thụng lng cao v chn lc amplicons ó 53 phỏt hin 16 ngnh vi khun liờn kt vi hi miờn R.globostellata Steinert v cs (2016) ó s dng pyrosequencing vựng V4-V5 ca gen 16S rRNA ó phỏt hin 18 ngnh vi khun liờn kt hi miờn R.globostellata v ngnh Archaea Crenarchaeota Cỏc OTUs chim u th gm cỏc thnh viờn ca Proteobacteria (Alpha-,Beta-, Delta-, and Gamma-), Cyanobacteria, Acidobacteria, Chloroflexi, ``Poribacteria'', Actinobacteria, Nitrospirae v PAUC34f Trong mu DN9, ch phỏt hin c 11 ngnh, v archaea l Thaumarchaeota Tuy nhiờn cỏc ngnh chim a s u ging nh cỏc nghiờn cu ca cỏc tỏc gi khỏc Theo chỳng tụi c bit ch cú nht nghiờn cu cu trỳc cng ng vi sinh vt ca hi miờn bin Dactylospongia metachromia thu thp ti Chuuk, Micronesia ca Jeong & Park (2013) Cỏc tỏc gi ó s dng in di trờn gel gradient bin tớnh (DGGE) gen 16S rRNA v ó phỏt hin c phyla, classes gm Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, Spirochaetes Phõn tớch phỏt sinh loi da trờn kt qu DGGE cho thy cng ng vi sinh vt ca mu hi miờn D metachromia cựng loi v cựng v trớ a lý ging Trong mu hi miờn DN12, 13 ngnh ó c phỏt hin vi cỏc ngnh ch o l Actinobacteria v Proteobacteria Kt qu phõn tớch a dng vi sinh vt liờn kt loi hi miờn ti bin Nng cho thy, chỳng cú s a dng tng i ging cỏc mc phõn loi, vi s phong phỳ ca tng ngnh, lp, b, h, chi khỏc Tuy nhiờn, mi loi hi miờn cng cha cỏc loi vi sinh vt c trng khụng phỏt hin c cỏc loi hi miờn khỏc Kt qu ny cng c ý kin cho rng, cng ng vi sinh vt liờn kt hi miờn chia s cỏc c tớnh quan trng gia cỏc loi hi miờn khỏc v cỏc cng ng c hiu hi miờn b nh hng bi vt ch [Jeong & Park, 2013; Steinert et al, 2016] Nghiờn cu cng ng vi khun liờn kt vi hai mu hi miờn Dactylospongia metachromia CH607 v CH840 c thu thp t qun o Chuuk thuc liờn bang Micronesia vo thỏng nm 2012 Bng phng phỏp DGGE, cỏc nh khoa hc Hn 54 Quc ó phỏt hin ngnh vi khun, lp: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, spirochaetes Da trờn phõn tớch phỏt sinh loi - DGGE fingerprint cho thy cu trỳc cng ng vi khun ging ht hai mu hi miờn cựng loi thu c t v trớ a lý ging Cú ti 93 ~ 100% trỡnh t t cỏc DGGE bands l tng ng vi cỏc loi vi khun c bit n c s d liu chung v s tng ng cao vi cỏc dũng vi khun khụng nuụi cy c 55 KT LUN ti c thc hin thụng qua s dng phng phỏp gii trỡnh t vựng V4 ca gen 16S rRNA trờn h thng MiSeq- Illumina ỏnh giỏ s a dng ca vi sinh vt liờn kt vi ba loi hi miờn Rhabdastrella globostellata (DN9); Cinachyrella sp (DN10); Dactylospongia sp (DN12) thu thp t vựng bin Nng, Vit Nam S dng khúa phõn loi Bayes v trỡnh t tham chiu t c s d liu Silva ó cho thy, vi sinh vt liờn kt vi Rhabdastrella globostellata (DN9) cú gii, 11 ngnh, 20 lp, 32 b, 18 h, chi (vi tng reads ln hn 0.1%) ú chi chớnh phỏt hin l: Caldilinea (21.45%); Nitrospira (4.55%) hi miờn Cinachyrella sp (DN10) ó tỡm thy gii, 12 ngnh, 21 lp, 29 b, 20 h v 17 chi (vi tng reads ln hn 0.1%), ú chi chớnh c phỏt hin l: Nitrospira; Nitrosococcus; Aeribacillus; Pseudoalteromonas; Halomonas mu Dactylospongia sp (DN12) phỏt hin gii, 13 ngnh, 22 lp, 36 b, 21 h, 10 chi (vi tng reads ln hn 0.1%) Cỏc chi chớnh c tỡm thy: Nitrospira; Caldilinea; Defluviicoccus; Endozoicomonas; Pseudovibrio Cng ng vi sinh vt liờn kt mu hi miờn nghiờn cu cú thnh phn c bn ging nhau, nhiờn mi mu li cú nhng thnh phn c trng cho loi m khụng phỏt hin c cỏc loi khỏc S khỏc bit ny ph thuc vo loi hi miờn khỏc Phỏt hin nhiu chi cú vai trũ quan trng i vi hi miờn v cú kh nng sinh cỏc cht cú hot tớnh sinh hc mu hi miờn kho sỏt (Aeribacillus; Halomonas; Nitrospira; Pseudovibrio; Ruegeria; Nitrosococcus; Bdellovibrio; ) 56 KIN NGH Vic nghiờn cu mi liờn h gia vi sinh vt liờn kt vi hi miờn thụng qua nghiờn cu metagenomics cú tớnh ng dng thc tin cao vic xỏc nh loi vi sinh vt mi; ỏnh giỏ a dng sinh hc vi sinh vt; xỏc nh thnh phn vi sinh vt mu mụi trng, c bit vi nhng mu khú/khụng th nuụi cy c iu kin phũng thớ nghim Quan trng hn, chỳng ta cú th phỏt hin nhiu loi vi sinh vt liờn kt vi hi miờn cú kh nng tng hp cỏc cht cú hot tớnh sinh hc cú ý ngha lnh vc y dc, hoỏ m phm Vi nhiu li ớch c th hin trờn, tụi xin cú ba kin ngh: Tip tc thc hin m rng cỏc nghiờn cu v a dng sinh hc vi sinh vt cú liờn kt vi hi miờn/ng vt bin khỏc ti 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(2007), In situ feeding and metabolism of glass sponges (Hexactinellida, Porifera) studied in a deep temperate fjord with a remotely operated submersible, Limnol Oceanogr 52, pp 428-440 63 Zhi-Yong Li *, Li-Ming He, Jie Wu, Qun Jiang(2005), Bacterial community diversity associated with four marine sponges from the South China Sea based on 16S rDNA-DGGE fingerprinting, Marine Biotechnology Laboratory, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China 66 64 https://www.boundless.com/biology/textbooks/boundless-biologytextbook/invertebrates-28/phylum-porifera-166/morphology-of-sponges-64211864/ https://sites.google.com/a/rsu35.org/seventh-grade-science/unit-6-aquaticecology 67 ... HỌC QUỐC GIA HÀ NỘI TRƢỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN - NGUYỄN MẠNH HÙNG ĐÁNH GIÁ ĐA DẠNG SINH HỌC VI SINH VẬT LIÊN KẾT VỚI HẢI MIÊN TẠI VÙNG BIỂN ĐÀ NẴNG VI T NAM THÔNG QUA NGHIÊN CỨU... kết với hải miên Bảng 3.8 Kết phân loại vi sinh vật liên kết với hải miên Bảng 3.9: Kết phân loại chi vi sinh vật liên kết với hải miên MỞ ĐẦU Hải miên vật chủ nhiều vi sinh vật vai trò vi sinh. .. kết với hải miên mức giới Bảng 3.5: Kết phân loại ngành vi sinh vật liên kết với hải miên Bảng 3.6 Kết phân loại lớp vi sinh vật liên kết với hải miên Bảng 3.7 Kết phân loại vi sinh vật liên kết