Đánh giá Đa dạng trình tự adn its của loài muồng ở việt nam

LI CM NVi lng knh trng v bit n su sc, em xin php c gi li cm n chn thnh nht ti :TS. Phng Thanh HngThs. H Th Thu Nhng ngi Thy trc tip hng dn tn tnh, du dt,gip v c bit lun truyn nhit huyt cho em trong sut qu trnh thc hin kha lun.XinchnthnhcmnccThy,Cgio,ccchkthutvin-bmnHasinh-trng i hc Dc H Ni to iu kin thun li gip em hon thnh ti.Xin cm n cc anh, ch ang cng tc ti Phng Vi sinh phn t - Vin Cng ngh sinh hc-VinHnlmKhoahc v Cng ngh Vit Nam ht lng gip em trong qu trnh thc hin ti tt nghip.Cuicngxincmngianh,bnblunlunlnglc,lnguncv,ng vin gip em hon thnh chng trnh i hc v kha lun tt nghip.

Sinh vin Phm Vit CngMC LCDANH MC T VIT TTDANH MC BNGDANH MC HNH NHT VN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Chng 1. TNG QUAN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1.Tng quan v vng phin m ni ca ADN ribosom (ITS-rADN) . . . . . . . . . . . 31.1.1.Cu trc ITS-rADN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1.2.Vai tr ca ITS-rADN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.1.3.ng dng ca ITS-rADN trong nghin cu cy thuc . . . . . . . . . . . . . . . . . . . . . . . 61.2.Tnh hnh nghin cu a dng di truyn cy thuc trn th gii v Vit Nam 81.2.1.Tnh hnh nghin cu a dng di truyn cy thuc trn th gii . . . . . . . . . . . . . . 81.2.2.Tnh hnh nghin cu a dng di truyn cy thuc Vit Nam. . . . . . . . . . . . . . . 91.3.Mung trung (Zanthoxylum avicennae (Lam.) DC.). . . . . . . . . . . . . . . . . . . . . . 101.3.1.Gii thiu v h Cam (Rutaceae) v chi Xuyn tiu (Zanthoxylum). . . . . . . . . 101.3.2.Loi Mung trung (Zanthoxylum avicennae (Lam.) DC.) . . . . . . . . . . . . . . . . . 111.3.3.Tnh hnh nghin cu v s dng loi Mung trung . . . . . . . . . . . . . . . . . . . . . . 13Chng 2.I TNG V PHNG PHP NGHIN CU . . . . . . . . . . . . . 152.1.i tng, nguyn liu nghin cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.1.1.i tng nghin cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.1.2.Ha cht, sinh phm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.1.3.Trang thit b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2.Phng php nghin cu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2.1.Phng php tch chit ADN tng s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.2.2.Phng php khuch i ADN bngPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.2.3.Phng php in di ADN trn gel agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.2.4.Phng php tch dng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.2.5.Phng php bin np ADN plasmid vo t bo kh bin E.coli . . . . . . . . . . . 222.2.6.Phng php tch chit ADN plasmid t vi khun. . . . . . . . . . . . . . . . . . . . . . . . . 242.2.7.Phng php ct ADN bng enzym gii hn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.2.8.Phng php tinh sch ADN plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252.2.9.Phng php gii trnh t ADN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.2.10.Phng php so snh trnh t ADN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Chng 3.THC NGHIM VKT QU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.1.Kt qu tch chit ADN tng s t l cy Mung trung .283.2.Kt qu khuch i ADN bng PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.3.Kt qu chn dng gen ITS-rADN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.4.Kt qu tinh sch plasmid ti t hp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.5. Kt qu gii trnh t vng ITS-rADN ca 5 mu Mung trung . . . . . . . . . 333.6.Kt qu so snh trnh t nucleotid vng ITS-rADN. . . . . . . . . . . . . . . . . . . . . . . 353.6.1.Kt qu so snh trnh t nucleotid vng ITS-rADN gia 5 mu Mung trung.353.6.2.Kt qu so snh trnh t nucleotid vng ITS-rADN gia 5 mu Mung trung vi 2 trnh t ca chi Zanthoxylum c cng b trn ngn hng gen th gii . . . . . . . . . . . . . . . . . 373.6.3.Kt qu cy phn loi trnh t ITS-rADN ca 5 mu Mung trung. . . . . . . . 38Chng 4. BN LUN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404.1. V quy trnh xc nh trnh t ITS-rADN ca loi Mung trung. . . . . . . . 404.2. V s a dng di truyn ca loi Mung trung Vit Nam . . . . . . . . . . . . . 41KT LUN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 XUT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44TI LIU THAM KHODANH MC T VIT TTA,T,G,C Adenin, Thymin, Guanin, CytosinADN Acid deoxyribonucleicrADN ADN m ha cho ARN ribosomAFLP Amplified Fragment Length PolymorphismATCC American Type Culture Collectionbp Base pairDMEM Dulbeccos Modified Eagles MediumDMSO DimethylsulfoxiddNTP Deoxyribonucleotid triphosphatE.coli Escherichia coliEDTA Ethylen Diamin Tetra acetic AcidEtBr Ethidium BromidHA22T T bo ung th biu m ganIGS Intergenic spacerIPTG Isopropyl -D-1- thiogalactopyranosidISSRInter-Simple Sequence RepeatITS Internal Transcribed Spacer (Vng phin m ni)ITS-rADN Internal Transcribed Spacer ribosomalAND (Trnh t ADN ribosom on ITS)K-562T bo ung th bch cu ngiKHTN Khoa hc t nhinLB Luria-BertaniOD Optical DensityPCR Polymerase Chain ReactionPP2A Protein phosphatase 2ARAPD Random Amplified Polymorphic DNASAMPL Selective Amplification ofMicrosatellite Polymorphic LociSDS Sodium Dodecyl SulphatTAE Tris - Acetic acid EDTATaq Thermus aquaticusX-gal 5-bromo-4-Chloro-3-Indolyl--D-GalactosidDANH MC BNGBng 1.1. Kch thc v phn trm G+C ca vng ITS1 v ITS2 ca mt s loi thc vt ht kn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Bng 2.1. Danh sch a im thu hi 5 mu Mung trung . . . . . . . . . . . . . . . . . . 15Bng 2.2. Cp mi dng trong phn ng PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Bng 2.3. Thnh phn phn ng PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Bng 2.4. Chu trnh nhit ca phn ng PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Bng 2.5. Thnh phn phn ng to vertor ti t hp . . . . . . . . . . . . . . . . . . . . . . . . 22Bng 2.6. Thnh phn phn ng ct plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Bng 2.7. Thnh phn phn ng gii trnh t gen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Bng 2.8. Chu trnh nhit phn ng c trnh t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Bng 3.1. Kt qu kim tra tinh sch v nng ADN tng s . . . . . . . . . . . . 29Bng 3.2. T l %(G+C) vng ITS-rADN ca 5 mu Mung trung . . . . . . . . . . 35Bng 3.3. H s tng ngtrnh t vng ITS-rADN gia 5 mu Mung trung37Bng 3.4. H s tng ng trnh t vng ITS-rADN gia 5 mu ZA1-ZA5 v mu ZAK, ZSK. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38DANH MC HNH NHHnh 1.1. Cu trc tng th ca vng ADN ribosom trong thc vt. . . . . . . . . . . . 3Hnh 1.2. Cu trc ca vng phin m ni ITS-rADN . . . . . . . . . . . . . . . . . . . . . . . . . . 4Hnh 1.3. Mung trung (Zanthoxylum avicennae (Lam.) DC.) . . . . . . . . . . . . . . . 12Hnh 2.1. S ni dung nghin cu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Hnh 3.1. Kt qu tch chit ADN tng s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Hnh 3.2. Sn phm PCR ca 5 mu Mung trung . . . . . . . . . . . . . . . . . . . . . . . . . . 29Hnh 3.3. nh in di th hin plasmid tch t t bo E.coli sau khi bin np ca cc mu ZA1 v ZA2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Hnh 3.4. nh in di th hin plasmid tch t t bo E.coli sau khi bin np ca cc mu ZA3, ZA4, ZA5.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Hnh 3.5. Sn phm ct plasmid bng enzym gii hn EcoRI . . . . . . . . . . . . . . . . . 32Hnh 3.6. Sn phm tinh sch plasmid mang gen ITS-rADN. . . . . . . . . . . . . . . . . . 33Hnh 3.7. Trnh t nucleotid on ITS-rADN ca 5 mu Mung trung. . . . . . 35Hnh 3.8. Kt qu so snh trnh t nucleotid vng ITS-rADN gia 5 mu Mung trung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Hnh 3.9.Cy phn loi5 mu Mung trung da trn so snh trnh t ITS-rADN 38T VN Vit Nam c nh gi l quc gia c ngun cy thuc t nhin phong ph v a dng vchngloilncngdnglmthuc.Tuynhinhinnaychckhong5%s loi c cng dng lm thuc c gii thiu v a vo khai thc, s loi cha c nghin cu v nh gi hincnrtln[3].Cngvinhucusdngcythuchngnmtrongncngycng tng,trongkhinguncythuc t nhin li cha c qun l khai thc cht ch dnn cn kitvslngvmtdntnhadng.Trongbicnhnhvycnnhanhchngthchin nhng bin php nhm bo v, s dng v pht trin ngun cy thuc mt cch hp l. Trong s nhngbinphp cn thit hin nay,nh gi a dng di truyn ngun cy thuc c vai tr c bitquantrng.Viclmnycnhiunghatrongnghincu,khaithcvsdngngun cythuc,nh:xydngvbotnngungen;chnto ging cho nng sut cao, cht lng tt;xcnhngungc,quanhtinha;nhndindcliumtcchchnhxc.Ngoira nhng d liu v cu trc gen ca cc qun th cy thuc cn cn thit cho chin lc khai thc v s dng hiu qu ngun ti nguyn cy thuc [14], [30]. nh gi a dng di truyn cy thuc, hin nay trn th gii c nhiu loi ch th ADN v marker phn t c s dng nh RAPD-PCR, AFLP, ITS-rADN, ADN lp th Trong s trnhtvngphinmnicaADNribosom(ITS-rADN) c bit n l marker phn t cnhiu u im ni tri trong nh gi a dng di truyn nh tnh cht va bo th, va siu bin c th gip xc nh ngun gc v quan h tin ha gia cc loi hoc gia cc c th trong cng mt loi, hay c lp li cao trong ADN nhn gip thc hin cc k thut khuch i, gii trnh t d dng hn [16], [40]. Mc d c ng dng rng ri trong nghin cu di truyn thc vt trn th gii nhng trnh t vng phin m ni ca ADN ribosom li hu nh cha c s dng trong nh gi a dng di truyn cy thuc Vit Nam.Mungtrung(Zanthoxylumavicennae(Lam.) DC.) l v thuc t lu c dn gian s dng cha bnh mn nga, l lot [10]. Gn y, mt s nghin cu trn th gii cho thy tc dng c ch s pht trin t bo ung th, tc dng khng khun ca dch chit t cy Mung trung[21],[43].Bncnhr,lcyMungtrungchanhiuhpchtthucccnhm: alkaloid(chimphnlnlberberin),terpenoid(tinhdu),flavanoidvcoumarin[5].Cth thy Mung trung l cy thuc c nhiu tim nng trong nghin cu sn xut dc phm. Tuy nhinnctahinchacnhiunghincuvloiMungtrung,nhngnghincuv Mung trung mi ch dng li vic xc nh thnh phn ha hc. Do vy cn y mnh nhiu nghin cu trn loi Mung trung hn na, trong bao gm cc nghin cu v di truyn, bo tn gen. Xut pht t nhng l do trn ti nhgiadngtrnhtADNITScaloiMungtrungVitNamc thc hin vi hai mc tiu sau:1.XcnhtrnhtvngITS-rADNca5muloiMungtrung(Zanthoxylum avicennae (Lam.) DC.) thu hi cc vng khc nhau ca Vit Nam.2.SosnhtrnhtvngITS-rADNgia5cthloiMungtrung(Zanthoxylum avicennae (Lam.) DC.) Vit Nam vi nhau v vi trnh t cng b trn ngn hng gen th gii, qua nh gi a dng di truyn loi Mung trung (Zanthoxylum avicennae (Lam.) DC.) Vit Nam.Chng 1. TNG QUANTng quan v vng phin m ni ca ADN ribosom (ITS-rADN)Cu trc ITS-rADNTrongcchuiADNnhncatbothcvt,khuvccnghincunhiunhtl vng ADN ribosom 18S-26S v hai vng phin m ni (ITS1, ITS2) vi nhiu on lp v trnh t nucleotid [15]. Di y l s th hin cu trc tng th ca ADN ribosom .Hnh 1.1.Cu trc tng th ca vng ADN ribosom trong thc vt [33]V tr ca ADN ribosom trn nhim sc th.Cc on gen (18S-5,8S-26S) xp lin tip nhau trn ADN ribosom.ADN ribosom gm nhng on gen (18S-5,8S-26S) c xp lin tip nhau, chia ct cc ongennyl1vnglinktIGS(intergenicspacer)ckchthcthayitrongccqun th khc nhau, c khi l gia cc c th [33]. Trong on gen (18S-5,8S-26S) vng ITS nm gia n v 18S v 26S. Vng ITS c 3 phn: ITS1; 5,8S v ITS2, trong tiu n v 5,8S l vng ctrnhtbotntin ha cao. Vng ITSc chng minh l trnh t ADN mang nhng c tnh hu ch cho vic nghin cu pht sinh loi ca thc vt ht kn [16]. Hnh 1.2. Cu trc ca vng phin m ni ITS-rADN [16]Trongmtnghincuthngkchothymtsloithcvtchoa,kchthcca vngITS1 v ITS2 thng nh hn 300bp (ITS1: 187-298 bp; ITS2: 187-252 bp), ngc li, mtssinhvtnhnthtkhckchthcca2vngnyc th ln hn nhiu, v d nh cc loingvtcxngsng.Vngtiuphn5,8Sckchthckhngil163-164bp. thc vt, kch thc ca ton b vng ITS xp x 500-750 bp thc vt ht kn v xp x 1500-3700bpthcvthttrn.Vd:mtnghincuchothykchthcvngITScachi Phyllanthus dao ng trong t 571-597 bp, loi Breynia fruticosa c kch thc vng ITS l 608 bp v loi Euphorbia milii l 649 bp. Kch thc vng ITS nm xp x 600-800 bp [15], [16], [29]. VngITS1thngckchthclnhnvngITS2,vdnhmtsloisau: Adoxaceae,Asteraceae,Brassicaceae,Canellaceae,Malvaceae,Onagraceae,Polemoniaceae. Ngcli,mtsloivngITS2ckchthclnhnvngITS1nh:Betulaceae, Cucurbitaceae, Scrophulariaceaev Viscaceae. loi Solanaceae, hai vng ny c kch thc gn nh bng nhau. i vi nhng h ln nh: Fabaceae, Poaceae v Rosaceae, vng ITS1 c kch thc di hn hoc ngn hn vng ITS2. Vng ITS1 v ITS2 khc nhau c v kch thuc v % (G+C). Sau y l mt v d v kch thc v % (G+C) ca 2 vngITS1 v ITS2 ca mt s loi [16]. Bng 1.1. Kch thc v phn trm G+C ca vng ITS1 v ITS2 ca mt s loi thc vt ht kn [16]NhmITS1 ITS2Nhm Kch thc(bp)%(G+C) Kch thc(bp)%(G+C)Adoxaceae, Viburnum, 28 loi 224-231 59-69215-22759-69Betulaceae,Alnu, 3 loi 215-216 58-64 228-229 53-65Canellaceae,Canella, 1 loi 272 63 209 63Fabaceae,Galegeae, 31 loi 221-231 55-60 207-217 50-54Poaceae,Pooideae,10 loi 217-223 55-64 213-221 59-67Vai tr ca ITS-rADNGing nh nhng sinh vt nhn tht khc, h gen thc vt bc cao bao gm ADN nhn v ADN ngoi nhn. ADN ngoi nhn gm c ADN lp th v ADN ti th. ADN ti th do ch mang ngun gc t m, khng nh gi chnh xc c bn cht di truyn nn rt t c dng trong nghin cu pht sinh loi. ADN lp th thc vt c tnh bo th cao v c tn s t bin thp do c dng lm cn c tm hiu ngun gc ca cc loi v trn loi. Tuy nhin tm hiu ngungcccbcphnloi thp hn ADN nhn t ra c nhiu u th bi ADN nhn c mc tinhanhanhhn,chophpphntchcskhcbitvditruyngiaccloitrong cng mt chi hay gia cc c th ca cng mt loi. Bn cnh c nhiu bng chng chng minhkhitihincutrcccloicquanhditruynvicsdngADNngoinhncth lm sai lch ln n s phn loi [15], [17].VngphinmnicaADNribosom(ITS-rADN) lvnggenvamangtnhboth (on 5,8S) va mang tnh siu bin (vng ITS1 v ITS2) do c kh nng phn nh quan h tinhacngnhnhgi a dng di truyn mc chi, loi v di loi. Tri li, trnh t nucleotid ca vng 18S v ca ADN lp th c mc bo th qu cao nn khng chi tit phn nh gia cc c th trong cng mt loi [16], [40]. Songsongvictnhhuchnitrn,trnhtvngphinmnicaADNribosom cn c mt s u im ni bt khng nh u th trong nghin cu pht sinh loi v nh gi a dngditruyncathcvthtkn,l:thnht,vngITSmangctnhditruyncac b v m, khc vi cc marker ADN lp th hay ADN ti th ch mang c tnh di truyn ca m. Th hai, lp li cao trong ADN ribosom ca thc vt, mi on ADN c sp xp lp lihngnghnbncopynhimscth,slngtrnhtlplinythunlichovicpht hin, khuch i v xc nh trnh t ADN ribosom. Th ba, trnh t vng ITS c tnh ph qut, v th cp mi dng cho phn ng khuchi c th d dng s dng trong cc nghin cu m vnchoktquchiu.uimthtlvngITSckchthcnh(thng