Định loại loài tảo Prorocentrum sp. phân lập được ở thành phố Hải Phòng dựa vào trình tự Nucleotit của các đoạn gien18s rDNA và ITS1-5,8S-ITS2

28(1): 81-91 T¹p chÝ Sinh häc 3-2006<br /> <br /> <br /> <br /> §Þnh lo¹i loµi t¶o PROROCENTRUM SP. ph©n lËp ®−îc<br /> ë thµnh phè H¶i phßng DùA VµO Tr×nh tù NUCLEOTIT<br /> cñA c¸c Đo¹n gien18S rDNA Vµ ITS1-5,8S-ITS2<br /> <br /> ®Æng DiÔm Hång, Hoµng Minh HiÒn, Hoµng Lan Anh<br /> ViÖn C«ng nghÖ sinh häc<br /> Chu V¨n Thuéc<br /> ViÖn Tµi nguyªn vµ M«i tr−êng biÓn<br /> <br /> Chi Prorocentrum thuéc ngµnh Dinoflagel- c¹nh c¸c ph−¬ng ph¸p ph©n lo¹i truyÒn thèng,<br /> latae bao gåm 31 loµi t¶o biÓn víi cÊu tróc tÕ bµo c¸c kü thuËt sinh häc ph©n tö ®d ®−îc sö dông<br /> rÊt gièng nhau [9]. Ng−êi ta thÊy chóng cã mÆt ë nh»m gãp phÇn ph©n lo¹i mét c¸ch chÝnh x¸c h¬n<br /> nh÷ng n¬i xuÊt hiÖn thuû triÒu ®á hoÆc cã sù “në c¸c loµi t¶o.<br /> hoa” cña c¶ t¶o ®éc vµ kh«ng ®éc. §éc tè DSP Trong bµi b¸o nµy, chóng t«i tr×nh bµy c¸c<br /> (Diarrhetic shellfish poisoning) cã trong c¸c loµi kÕt qu¶ b−íc ®Çu ph©n lo¹i loµi t¶o<br /> t¶o gi¸p sèng tr«i næi hoÆc sèng ®¸y, hÇu hÕt Prorocentrum sp. thu ®−îc t¹i thµnh phè H¶i<br /> thuéc chi Dinophysis hoÆc chi Prorocentrum, Phßng b»ng ph−¬ng ph¸p dùa vµo c¸c ®Æc ®iÓm<br /> g©y ¶nh h−ëng ®Õn hÖ tiªu hãa cña ng−êi. HiÖn h×nh th¸i kÕt hîp víi so s¸nh tr×nh tù nucleotit<br /> t−îng “në hoa” cña c¸c loµi t¶o nµy trªn biÓn vµ cña c¸c ®o¹n gien 18S rDNA, ITS1-5,8S-ITS2.<br /> ë c¸c thñy vùc th−êng kÐo theo sù nhiÔm ®éc cho Trªn c¬ së c¸c kÕt qu¶ thu ®−îc, tªn vµ mèi<br /> c¸c loµi h¶i s¶n vµ con ng−êi khi ¨n ph¶i chóng. quan hÖ vÒ ph¸t sinh chñng lo¹i gi÷a loµi mµ<br /> Do vËy, viÖc ph¸t hiÖn vµ ng¨n chÆn sù në hoa chóng t«i ph©n lËp ®−îc víi c¸c loµi<br /> cña t¶o ®éc lµ rÊt cÇn thiÕt vµ cã ý nghÜa thùc tiÔn Prorocentrum spp. kh¸c ®d ®−îc c«ng bè t¹i<br /> to lín trong viÖc gi¶m thiÓu nh÷ng t¸c ®éng xÊu Ng©n hµng gien thÕ giíi GENEBANK.<br /> cña chóng tíi m«i tr−êng, ®Æc biÖt lµ ®èi víi c«ng<br /> viÖc nu«i trång h¶i s¶n vµ b¶o ®¶m an toµn thùc I. Ph−¬ng ph¸p nghiªn cøu<br /> phÈm [6, 9]. C¸c nghiªn cøu vÒ sù “në hoa” cña<br /> t¶o ®éc bao gåm viÖc x¸c ®Þnh vµ thèng kª c¸c 1. VËt liÖu<br /> loµi t¶o g©y ®éc vµ h¹i, sù ph©n bè cña chóng<br /> trong kh«ng gian, thêi gian vµ c¸c yÕu tè m«i - VËt mÉu Prorocentrum sp. ®−îc ph©n lËp ë<br /> tr−êng liªn quan ®Õn hiÖn t−îng bïng ph¸t sè §å S¬n, thµnh phè H¶i Phßng do ViÖn Tµi<br /> l−îng t¶o ®éc [1] vµ c¸c biÖn ph¸p phßng ngõa nguyªn vµ M«i tr−êng biÓn cung cÊp. §é thuÇn<br /> gi¶m thiÓu t¸c h¹i cña chóng. Ph−¬ng ph¸p ph©n khiÕt theo tiªu chuÈn h×nh th¸i cña t¶o ®−îc<br /> lo¹i truyÒn thèng dùa trªn c¸c ®Æc ®iÓm h×nh th¸i kiÓm tra d−íi kÝnh hiÓn vi laser quÐt Axiovert<br /> cã vai trß quan träng trong viÖc x¸c ®Þnh c¸c loµi 100M cña hdng CarlZeiis.<br /> t¶o, tuy nhiªn, cã rÊt nhiÒu khã kh¨n khi gÆp - C¸c tr×nh tù cña c¸c ®o¹n gien 18S rDNA<br /> nh÷ng loµi cã kh¶ n¨ng biÕn ®æi h×nh th¸i ®Ó vµ ITS1-5,8S-ITS2 cña 10 loài Prorocentrum ®d<br /> thÝch øng trong nh÷ng ®iÒu kiÖn sèng kh¸c nhau ®−îc c«ng bè t¹i Ng©n hàng gien GENEBANK<br /> hoÆc nh÷ng loµi rÊt gièng nhau vÒ mÆt h×nh th¸i (b¶ng 1) ®d ®−îc sö dông.<br /> vµ nh÷ng biÕn th¸i nµy cña chóng l¹i rÊt khã<br /> ph©n biÖt d−íi kÝnh hiÓn vi [6, 7]. HiÖn nay, bªn - Vect¬ pCRR2.1, chñng vi khuÈn E.coli<br /> <br /> <br /> <br /> C«ng tr×nh ®−îc sù hç trî cña Ch−¬ng tr×nh khoa häc KC.09.19 vµ Ch−¬ng tr×nh ®iÒu tra nghiªn cøu øng<br /> dông c«ng nghÖ biÓn.<br /> 81<br /> DH5α và c¸c hãa chÊt chuÈn cña hdng theo nh− c«ng bè [4, 5].<br /> Invitrogen. - Qu¸ tr×nh t¸ch dßng c¸c ®o¹n gien nh©n<br /> 2. Ph−¬ng ph¸p ®−îc, ®−îc tiÕn hµnh theo c«ng bè [4, 5, 8].<br /> - Tr×nh tù nucleotit cña c¸c ®o¹n gien 18S<br /> - T¸ch chiÕt DNA tæng sè cña Prorocentrum rDNA và ITS1-5,8S-ITS2 ë mÉu nghiªn cøu ®−îc<br /> sp. theo ph−¬ng ph¸p ®d c«ng bè [5]. thùc hiÖn trªn m¸y ®äc tr×nh tù tù ®éng ABI<br /> - C¸c ®o¹n gien 18S rDNA vµ ITS1-5,8S-ITS2 PRISM(R)3100-Avant Genetic Analyzer (ABI, Mü)<br /> ®−îc nh©n b»ng kü thuËt PCR tõ DNA tæng sè víi cña ViÖn C«ng nghÖ sinh häc.<br /> c¸c cÆp måi ®Æc hiÖu: 18S-F (5’-GAGAGGG- - Dùa trªn ch−¬ng tr×nh Clustal X Multiple<br /> AGCCTGAGAAACG-3’) vµ 18S-R (5’-GGCAT- Sequence Alignment Program (version 1.81,<br /> CACAGACCTGTTATTGC-3’), ITS-F (5’-TCCG- June 2000) vµ DNASTAR, chóng t«i x©y dùng<br /> TAGGTGAACCTGCGG-3’) vµ ITS-R (5’-CGA- c©y ph¸t sinh chñng lo¹i cña loµi Prorocentrum<br /> CGCAAGAAGTAGACTCG-3’). sp. thu thËp t¹i H¶i Phßng víi tr×nh tù cña c¸c<br /> - §iÒu kiÖn và thành phÇn cña ph¶n øng PCR loµi Prorocentrum cã s½n t¹i GENEBANK.<br /> B¶ng 1<br /> C¸c loài Prorocentrum cã tr×nh tù cña c¸c ®o¹n gien 18S rDNA và ITS1-5,8S-ITS2<br /> ®−îc sö dông ®Ó ph©n tÝch sù ®a d¹ng di truyÒn<br /> Số hiệu taxon M· sè<br /> STT Tªn khoa häc Gien m· hãa<br /> (TaxID) GENEBANK<br /> 1 Prorocentrum lima (Ehrenberg) Dodge 1975 39448 Y16235 18S rDNA<br /> 2 P. arenarium Faust 1994 72679 Y16234 18S rDNA<br /> 3 P. maculosum Faust 1993 72680 Y16236 18S rDNA<br /> 4 P. concavum Fukuyo 1981 72681 Y16237 18S rDNA<br /> 5 P. panamensis sp. nov. 72678 Y16233 18S rDNA<br /> 6 P. micans Ehrenberg 1834 2945 AJ415519 18S rDNA<br /> 7 P. minimum var. triangulatum Hulburt 1959 39449 AJ415520 18S rDNA<br /> 8 P. minimum (Pavillard) Schiller 1933 39449 Y16238 18S rDNA<br /> 9 P. mexicanum Osorio Tafall 1942 72677 Y16232 18S rDNA<br /> 10 P. emarginatum Fukuyo 1981 72682 Y16239 18S rDNA<br /> 11 P. micans Ehrenberg 1834 2945 AF208245 ITS1-5,8S-ITS2<br /> 12 P. minimum (Pavillard) Schiller 1933 39449 AF352370 ITS1-5,8S-ITS2<br /> 13 P. minimum var. triangulatum Hulburt 1959 39449 AF208244 ITS1-5,8S-ITS2<br /> 14 P. triestinum Schiller 1918 39450 AF208246 ITS1-5,8S-ITS2<br /> 15 P. minimum var. mariae-lebourae Hulburt 1959 39449 AF352371 ITS1-5,8S-ITS2<br /> <br /> II. KÕt qu¶ nghiªn cøu<br /> <br /> 1. M« t¶ h×nh th¸i cña loµi Prorocentrum sp.<br /> ®−îc ph©n lËp t¹i H¶i Phßng<br /> D−íi kÝnh hiÓn vi lazer quÐt, Prorocentrum<br /> sp. lµ c¸c ®¬n bµo chuyÓn ®éng; tÕ bµo gÇn<br /> gièng h×nh ovan, dài 32,58-35,33 µm, réng<br /> 22,84-23,66 µm. Nh− vËy, dùa vµo c¸c ®Æc ®iÓm<br /> h×nh th¸i, vËt mÉu Prorocentrum sp. mµ chóng<br /> t«i ph©n lËp ®−îc t¹i H¶i Phßng cã thÓ ®−îc xÕp<br /> vµo loµi Prorocentrum mexicanum Osorio Tafall H×nh 1. H×nh th¸i tÕ bào cña loài Prorocentrum<br /> 1942. sp. thu t¹i H¶i Phßng d−íi kÝnh hiÓn vi lazer quét.<br /> 82<br /> 2. T¸ch chiÕt DNA tæng sè 1,0 kb ®èi víi cÆp måi 18S F-R và 0,6 kb víi cÆp<br /> måi ITS F-R. S¶n phÈm PCR cña Prorocentrum<br /> KÕt qu¶ t¸ch DNA tæng sè cña Prorocentrum<br /> sp. víi hai cÆp måi nãi trªn ®−îc chØ ra trªn h×nh<br /> sp. ®−îc tr×nh bµy trªn h×nh 2 cho thÊy DNA cã<br /> 3. KÕt qu¶ cho thÊy chóng t«i ®d nh©n ®−îc c¸c<br /> chÊt l−îng tèt, kh«ng bÞ ®øt gdy, phï hîp ®Ó lµm ®o¹n gien 18S rDNA và ITS1-5,8S-ITS2 cã kÝch<br /> nguyªn liÖu cho c¸c nghiªn cøu tiÕp theo.<br /> th−íc t−¬ng øng kho¶ng 1,0 kp và 0,6 kp. KÝch<br /> M 1 2 3 4 th−íc cña c¸c s¶n phÈm PCR mµ chóng t«i thu<br /> ®−îc phï hîp víi kho¶ng c¸ch gi÷a hai måi vµ<br /> kÝch th−íc theo tÝnh to¸n lý thuyÕt.<br /> 4. T¸ch dßng c¸c ®o¹n gien 18S rDNA và<br /> ITS1-5,8S-ITS2<br /> 1 2 3 4 5 6 7 M<br /> <br /> <br /> <br /> <br /> H×nh 2. ¶nh ®iÖn di kiÓm tra DNA cña<br /> Prorocentrum sp. 1,0 kb<br /> GiÕng 1: mack¬ 1Kb; giÕng 1-4: mÉu DNA t¸ch tõ 0,6 kb<br /> Prorocentrum sp.<br /> 3. Nh©n c¸c ®o¹n gien 18S rDNA và ITS1-<br /> 5,8S-ITS2 b»ng kü thuËt PCR<br /> 1 2 M H×nh 4. §iÖn di s¶n phÈm PCR kiÓm tra c¸c<br /> ®o¹n gien 18S rDNA và ITS1-5,8S-ITS2 víi cÆp<br /> måi 18S R-F và ITS F-R ®d ®−îc g¾n vµo vect¬<br /> t¸ch dßng pCRR2.1.<br /> Cét M: thang chuÈn cña DNA cã kÝch th−íc 1<br /> kb; cét 1-4: c¸c dßng tÕ bµo mang vect¬ t¸i tæ hîp<br /> 1,0 kb g¾n ®o¹n gien 18S rDNA; cét 5-7: c¸c dßng tÕ bµo<br /> mang vect¬ t¸i tæ hîp g¾n ®o¹n gien ITS1-5,8S-ITS2.<br /> 0,6 kb<br /> 1 2 3 4 5 6 7 M<br /> <br /> <br /> <br /> H×nh 3. §iÖn di s¶n phÈm PCR cña loài<br /> Prorocentrum sp.<br /> Cét M: thang chuÈn cña DNA cã kÝch th−íc 1<br /> kb; cét 1: s¶n phÈm PCR víi cÆp måi 18S F-R; cét 2: 1,0 kb<br /> s¶n phÈm PCR víi cÆp måi ITS F-R. 0,6 kb<br /> §Ó ph©n lËp vµ t¸ch dßng mét phÇn cña c¸c<br /> ®o¹n gien 18S rDNA và ITS1-5,8S-ITS2 cña<br /> Prorocentrum sp., chóng t«i ®d thiÕt kÕ c¸c cÆp H×nh 5. Ph©n tÝch enzym giíi h¹n c¸c plasmit<br /> måi ®Æc hiÖu 18S F-R và ITS F-R dùa vào tr×nh t¸i tæ hîp mang c¸c ®o¹n gien 18S rDNA và<br /> tù nucleotit cña c¸c ®o¹n gien 18S rDNA và ITS1-5,8S-ITS2.<br /> ITS1-5,8S-ITS2 cña chi Prorocentrum ®d ®−îc Cét M: thang chuÈn DNA cã kÝch th−íc 1 kb; cét 1-<br /> c«ng bè t¹i Ng©n hµng gien quèc tÕ 3: vect¬ pCRR2.1 ®d g¾n s¶n phÈm PCR cña ®o¹n<br /> (GENEBANK). Trong ®ã, theo tÝnh to¸n lý gien18S rDNA; cét 4-7: vect¬ pCRR2.1 ®d g¾n s¶n phÈm<br /> thuyÕt, s¶n phÈm PCR sÏ cã kÝch th−íc kho¶ng PCR cña ®o¹n gien ITS1-5,8S-ITS2.<br /> 83<br />  C¸c ®o¹n gien nhËn ®−îc ®d ®−îc tiÕn hµnh trËn tam gi¸c t¹i c¸c b¶ng 2 vµ 3. Theo kÕt qu¶ ë<br /> t¸ch dßng, x¸c ®Þnh tr×nh tù theo c¸c c«ng bè [3, c¸c b¶ng 2 vµ 3, chóng t«i thÊy ®é t−¬ng ®ång cña<br /> 4, 7]. §Ó kh¼ng ®Þnh cã ph¶i lµ vect¬ t¸i tæ hîp ®o¹n gien 18S rDNA gi÷a c¸c loµi Prorocentrum lµ<br /> mang c¸c ®o¹n gien 18S rDNA và ITS1-5,8S- tõ 92-100% vµ cña ®o¹n gien ITS1-5,8S-ITS2 lµ tõ<br /> ITS2 mong muèn kh«ng, chóng t«i tiÕn hµnh 77-99%. Nh− vËy, viÖc ®äc vµ so s¸nh c¸c tr×nh tù<br /> ph¶n øng PCR-checking c¸c khuÈn l¹c tr¾ng víi cña c¸c ®o¹n gien 18S rDNA vµ ITS1-5,8S-ITS2<br /> hai cÆp måi 18S F-R và ITS F-R (h×nh 4) và c¾t trong chi Prorocentrum lµ c«ng cô rÊt h÷u hiÖu ®Ó<br /> DNA plasmit t¸i tæ hîp víi enzym giíi h¹n kiÓm tra vµ x¸c ®Þnh mèi quan hÖ ph¸t sinh chñng<br /> EcoRI (h×nh 5). KÕt qu¶ trªn c¸c h×nh 4, 5 thu lo¹i trong cïng mét loµi vµ d−íi loµi ë nh÷ng ®iÒu<br /> ®−îc ®d chøng tá r»ng c¸c ®o¹n gien mµ chóng kiÖn ®Þa lý, sinh th¸i kh¸c nhau. §iÒu nµy ®−îc chØ<br /> t«i mong muèn ®d ®−îc g¾n thµnh c«ng vµo ra khi so s¸nh 2 nhãm P. minimum víi nhau, ®é<br /> vect¬ t¸ch dßng pCRR2.1. TiÕp theo, c¸c dßng t−¬ng ®ång cña ®o¹n gien 18S rDNA gi÷a hai<br /> plasmit t¸i tæ hîp nãi trªn ®−îc t¸ch chiÕt vµ nhãm P. minimum là 100% (b¶ng 2) nh−ng víi<br /> tinh s¹ch mét l−îng lín ®Ó ®äc tr×nh tù. ®o¹n gien ITS1-5,8S-ITS2 là 99,3-99,5% (b¶ng 3).<br /> 5. So s¸nh tr×nh tù nucleotit cña c¸c ®o¹n gien Còng trªn b¶ng 2, chóng ta nhËn thÊy r»ng<br /> Prorocentrum sp. cã ®é t−¬ng ®ång cao nhÊt, ®¹t<br /> 18S rDNA và ITS1-5,8S-ITS2 cña c¸c loµi<br /> Prorocentrum ®Õn 99,9% khi so s¸nh víi P. mexicanum<br /> (Y16232), tiÕp ®ã 99,8% víi P. micans<br /> Chóng t«i ®d tiÕn hµnh so s¸nh c¸c tr×nh tù thu (AJ415519), 99,6% víi P. minimum (AJ415520 và<br /> ®−îc cña Prorocentrum sp. víi 10 tr×nh tù cña ®o¹n Y16238), 96,5% víi P. concavum (Y16237),<br /> gien 18S rDNA cña 9 loµi Prorocentrum kh¸c (h×nh 96,4% víi P. panamensis (Y16233), 95,6% víi P.<br /> 6) vµ víi 5 tr×nh tù cña ®o¹n gien ITS1-5,8S-ITS2 emarginatum (Y16239) và thÊp nhÊt là 94,1% víi<br /> cña 3 loµi Prorocentrum kh¸c ®d ®−îc c«ng bè t¹i P. arenarium (Y16234) và P. maculosum<br /> Ng©n hµng gien quèc tÕ (h×nh 7). KÕt qu¶ ë c¸c (Y16236). KÕt hîp víi c¸c ®Æc ®iÓm h×nh th¸i cña<br /> h×nh 6 vµ 7 cho thÊy cã sù sai kh¸c cña c¸c tr×nh tù Prorocentrum sp. miªu t¶ ë phÇn II.1 víi kÕt qu¶<br /> nµy gi÷a c¸c loµi Prorocentrum víi nhau. Tû lÖ ph©n tÝch tr×nh tù cña ®o¹n gien 18S rDNA trªn<br /> phÇn tr¨m t−¬ng ®ång cña tõng cÆp tr×nh tù cña c¸c ®©y, chóng t«i kÕt luËn r»ng Prorocentrum sp.<br /> loµi Prorocentrum ®−îc thèng kª d−íi d¹ng ma thuéc loµi Prorocentrum mexicanum.<br /> AJ415520 GAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16238 GAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16232 GAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Prorocentrum sp. GAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> AJ415519 GAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16239 GAGAGGGAGCCCGAGACATGGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16233 GAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGTAAATTACC<br /> Y16237 GAGAGGGAGCCTGAGAAATAGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16234 GAGAGGGAGCCTGAGAAATAGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16235 GAGAGGGAGCCTGAGAAATAGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> Y16236 GAGAGGGAGCCTGAGAAATAGCTACCACATCTAAGGAAGGCAGCAGGCGCGCAAATTACC<br /> *********** **** * ******************************* ********<br /> AJ415520 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATAATTGTCTTGTA<br /> Y16238 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATAATTGTCTTGTA<br /> Y16232 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATATCTGTCTTGTA<br /> Prorocentrum sp. CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATATCTGTCTTGTA<br /> AJ415519 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATATCTGTCTTGTA<br /> Y16239 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACACGGCATATTTGTCTTGTA<br /> Y16233 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAAGGCATCCATGTCTTGTA<br /> Y16237 CAATCCTGACATAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATTCTTGTCTTGTA<br /> Y16234 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATCCATGTCTTGTA<br /> Y16235 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATCCATGTCTTGTA<br /> Y16236 CAATCCTGACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCATCCATGTCTTGTA<br /> *********** ****************************** ***** *********<br /> AJ415520 ATTGGAATGAGTAGAATTTAAATCCCTTTACGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Y16238 ATTGGAATGAGTAGAATTTAAATCCCTTTACGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Y16232 ATTGGAATGAGTAGAATTTAAATCCCTTTACGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Prorocentrum sp. ATTGGAATGAGTAGAATTTAAATCCCTTTACGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> AJ415519 ATTGGAATGAGTAGAATTTAAATCCCTTTACGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> <br /> 84<br /> Y16239 ATTGGAATGAGTAGAATTTAAATCCCTTTACGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Y16233 ATTGGAATGAGTAGAACTCAAACTCCTCTACAAGTACCGATTGGAGGGCAAGTCTGGTGC<br /> Y16237 ATTGGAATGAGTAGAACTTAAATCCCTTTGCGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Y16234 ATTGGAATGAGTAGAACTTAAATCTCTTTGTGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Y16235 ATTGGAATGAGTAGAACTTAAATCTCTTTGTGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> Y16236 ATTGGAATGAGTAGAATTTAAATCTCTTTATGAGTACCAATTGGAGGGCAAGTCTGGTGC<br /> **************** * *** ** * ****** *********************<br /> AJ415520 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16238 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16232 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Prorocentrum sp. CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> AJ415519 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16239 CAGCAGCCGCGGTAATTCCAGCTCCAATGGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16233 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16237 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16234 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCATATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16235 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCATATATTAAAGTTGTTGCGGTTAAAAAGC<br /> Y16236 CAGCAGCCGCGGTAATTCCAGCTCCAATAGCATATATTAAAGTTGTTGCGGTTAAAAAGC<br /> **************************** ** ****************************<br /> AJ415520 TCGTAGTTGGATTTCTGCCGAGGACGACCGGTCCGCCCTCTGGGTGAGTATCTGGCTCGG<br /> Y16238 TCGTAGTTGGATTTCTGCCGAGGACGACCGGTCCGCCCTCTGGGTGAGTATCTGGCTCGG<br /> Y16232 TCGTAGTTGGATTTCTGCCGAGGACGACCGGTCCGCCCTCTGGGTGAGTATCTGGCTCGG<br /> Prorocentrum sp. TCGTAGTTGGATTTCTGCCGAGGACGACCGGTCCGCCCTCTGGGTGAGTATCTGGCTCGG<br /> AJ415519 TCGTAGTTGGATTTCTGCCGAGGACGACCGGTCCGCC-TCTGGGTGAGTATCTGGCTCGG<br /> Y16239 TCGTAGTTGGAAGTCTGCCTAGGAAGACTGGTCCGCCCTCCGGGTGAGTATCTGGCTCGG<br /> Y16233 TCGTAGTTGGACTTCTGCTGAGGACGACCGGTCCGCCCTCTGGGTGAGCATCTGGCTCGG<br /> Y16237 TCGTAGTCGGATTTCTGCCGAGGACGACCGGTCCGCCCTCTGGGTGAGTATCTGGCTCGG<br /> Y16234 TCGTAGTCGGATTTCTGCCGAGGACAACCGGTCCGCCCTCTGGGTGAGCATCTGGCTTGA<br /> Y16235 TCGTAGTCGGATTTCTGCCGAGGACAACCGGTCCGCCCTCTGGGTGAGCATCTGGCTTGA<br /> Y16236 TCGTAGTCGGATTTCTGCCGAGGACAACCGGTCCGCCCTCTGGGCGAGCATCTGGCTTGA<br /> ******* *** ***** **** ** ******** ** *** *** ******** *<br /> AJ415520 CCTGGGCATCTTCTTGGAGAACGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> Y16238 CCTGGGCATCTTCTTGGAGAACGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> Y16232 CCTGGGCATCTTCTTGGAGAACGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> Prorocentrum sp. CCTGGGCATCTTCTTGGAGAACGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> AJ415519 CCTGGGCATCTTCTTGGAGAACGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> Y16239 CCTGGGCATTTTCTTGGAGAATGTAGGTGCACTTGGCTGTGTGGTGCGGTATCCAGGACC<br /> Y16233 CCTTGGCATCTTCTTGGAGAACGCAACTGCACTTGACTGTGTGGTGCGGCATCCAGGACT<br /> Y16237 CCTGGGCATCTTCTTGGAAAGCGTGGCTGCACTTGATTGTGTGGCGCGGTAGCCAGGGCT<br /> Y16234 TCTGGGCATCTTCTTGGAGAGCGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> Y16235 TCTGGGCATCTTCTTGGAGAGCGTA-CTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> Y16236 TCTGGGCATCTTCTTGGAGAGCGTAGCTGCACTTGACTGTGTGGTGCGGTATCCAGGACT<br /> ** ***** ******** * * ******** ******* **** * ***** *<br /> AJ415520 TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCTTACGCCTTGAATACATTAGCATGG<br /> Y16238 TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCTTACGCCTTGAATACATTAGCATGG<br /> Y16232 TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCTTACGCCTTGAATACATTAGCATGG<br /> Prorocentrum sp. TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCTTACGCCTTGAATACATTAGCATGG<br /> AJ415519 TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCTTACGCCTTGAATACATTAGCATGG<br /> Y16239 TGTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCTGATGCCTTGTATACGTTAGCATGG<br /> Y16233 TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCACACGCTTTGAATACATTAGCATGG<br /> Y16237 TTTACTTTGAGGAAATTAGAGTGTTTCAAGCAGGCGCATGCTTTGAATACTTTAGCATGG<br /> Y16234 TTTACTTTGAGGAAATTAGAGTGTCCTAAGCAGGCCCATGCCATAAATACATTAGCATGG<br /> Y16235 TTTACTTTGAGGAAATTAGAGTGTCCTAAGCAGGCCCATGCCATATATACATTAGCATGG<br /> Y16236 TTTACTTTGAGGAAATTAGAGTGTCCCAAGCAGGCCCATGCCATAAATACGTTAGCATGG<br /> * ********************** ******** * ** * **** *********<br /> AJ415520 AATAATAAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGAGCTGAGG-TAATGATTA<br /> Y16238 AATAATAAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGAGCTGAGG-TAATGATTA<br /> Y16232 AATAATAAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGAGCTGAGG-TAATGATTA<br /> Prorocentrum sp. AATAATAAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGAGCTGAAG-TAATGATTA<br /> AJ415519 AATAATAAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGAGCTGAGG-TAATGATTA<br /> Y16239 CATACTAAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGAGCTGAGG-TAATGGTTA<br /> Y16233 AATAATAAGATAGGACCTTGGTTCTATTTTGTTGGTTTCTAGAGCCGAGGGTAATGATTG<br /> Y16237 AATAATAAGATAGGACCTCTGTTCTATTTTGTTGGTTTCTAGAGCAGAGG-TAATGATTA<br /> Y16234 AATAATAAGGTAGGACCTACTCTCTATTTTGTTGGTTTCTAGAGCAGAGG-TAATGGTCA<br /> Y16235 AATAATAAGGTAGGACCTACTCTCTATTTTGTTGGTTTCTAGAGCA-AGG-TAATGGTCA<br /> Y16236 AATAATAGGGTAGGACCTTTTCTCTATTTTGTTGGTTTCTAGAGCAGAGG-TAATGGTCA<br /> *** ** * ******** *********************** * * ***** *<br /> <br /> <br /> 85<br />  AJ415520 ATAGGGATAGTTGGGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Y16238 ATAGGGATAGTTGGGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Y16232 ATAGGGATAGTTGGGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Prorocentrum sp. ATAGGGATAGTTGGGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> AJ415519 ATAGGGATAGTTGGGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Y16239 ATAGGGATAGTTGGGGGTGTTCGTACTTAATTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Y16233 ATAGGGATAGTTGGGGGCATTCGAATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Y16237 ATAGGGATAGTTGGGGGCACTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTA<br /> Y16234 ATGGGGATAGTTGGGGGTATTCGTATTTGACTGTCAGAGGTGAAATTCTTGGATTTGTCA<br /> Y16235 ATGGGGATAGTTGGGGGTATTCGTATTTGACTGTCAGAGGTGAAATTCTTGGATTTGTCA<br /> Y16236 ATGGGGATAGTTGGGGGTATTCGTATTTGACTGTCAGAGGTGAAATTCTTGGATTTGTCA<br /> ** ************** *** * ** * *************************** *<br /> AJ415520 AAGACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCAAGAACGAAAGTT<br /> Y16238 AAGACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCAAGAACGAAAGTT<br /> Y16232 AAGACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCAAGAACGAAAGTT<br /> Prorocentrum sp. AAGACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCAAGAACGAAAGTT<br /> AJ415519 AAGACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCAAGAACGAAAGTT<br /> Y16239 AAGACGGACAACTGCGAAAGCATTCGCCAAGGATGTTCTCATTGATCAAGAACGAAAGTT<br /> Y16233 AAGATGGACTACTGCGAAAGCATTTGCCAAAGATGTTTTCATTGATCAAGAACGAAAGTT<br /> Y16237 AAGACGAACTACTGCGAAAGCATTTGCCAGGGATGTTTTCATTGATCAAGAACGAAAGTT<br /> Y16234 AAGACGAACCAATGCGAAAGCATTTGCCAGAGATGTTTTCCTTGATCAAGAACGAAAGTT<br /> Y16235 AAGACGAACCAATGCGAAAGCATTT-CCAGAGATGTTTTCCTTGATCAAGAACGAAAGTT<br /> Y16236 AAGACGAACCAATGCGAAAGCATTTGCCAGAGATGTTTTCCTTGATCAAGAACGAAAGTT<br /> **** * ** * ************ *** ****** ** *******************<br /> AJ415520 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> Y16238 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> Y16232 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> Prorocentrum sp. AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> AJ415519 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> Y16239 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> Y16233 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACCATGCCGACTAGAG<br /> Y16237 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACAATGCCAACTAGAG<br /> Y16234 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACTATGCCAACTAGAG<br /> Y16235 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACTATGCCAACTAGAG<br /> Y16236 AGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATAAACTATGCCAACTAGAG<br /> ********************************************** ***** *******<br /> AJ415520 ATTGGAGGTCGTTATTTATACGACTCCTTCAGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Y16238 ATTGGAGGTCGTTATTTATACGACTCCTTCAGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Y16232 ATTGGAGGTCGTTATCTATACGACTCCTTCAGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Prorocentrum sp. ATTGGAGGTCGTTATCTATACGACTCCTTCAGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> AJ415519 ATTGGAGGTCGTTATCTATACGACTCCTTCAGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Y16239 ATTGGAGGCCGTTATCTATGTGGCTCCTTCAGCACCTTATGAGAAATCAAAGTATTTGGG<br /> Y16233 ATTGGAGGTTGTTATCTTCTCGACTCCTCCAGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Y16237 ATTGGAGGTCGTTAGCTATACGACTCCTTCAGCACCTTATGAGAAATCAAAGTCCTTGGG<br /> Y16234 ATTGGAGGTCGTTATGTTGACGACTCTTTCGGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Y16235 ATTGGAGGT-GTTATGTTGACGACTCTTTCGGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> Y16236 ATTGGAGGTCGTTAGATTTACGACTCTTTCGGCACCTTATGAGAAATCAAAGTCTTTGGG<br /> ******** **** * * *** * * ********************** *****<br /> AJ415520 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16238 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16232 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Prorocentrum sp. TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> AJ415519 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16239 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16233 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16237 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16234 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16235 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> Y16236 TTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACC<br /> ************************************************************<br /> AJ415520 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCAGACA<br /> Y16238 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGGAAACTTACCAGGTCCAGACA<br /> Y16232 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGGAAACTTACCAGGTCCAGACA<br /> Prorocentrum sp. AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCAGACA<br /> AJ415519 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCAGACA<br /> Y16239 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCAGACA<br /> Y16233 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCGGACA<br /> Y16237 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCAGACA<br /> <br /> 86<br />  Y16234 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCGGACA<br /> Y16235 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCGGACA<br /> Y16236 AGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGG-AAACTTACCAGGTCCGGACA<br /> *************************************** *************** ****<br /> AJ415520 TAGTAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16238 TAGTAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16232 TAGTAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Prorocentrum sp. TAGTAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> AJ415519 TAGTAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16239 TAGTTAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16233 TAGTAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16237 TAGTAAGGATGGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16234 TAGTAAGGATTGACAGATTGACAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16235 TAGTAAGGATTGACAGATTGACAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> Y16236 TAGTAAGGATTGACAGATTGACAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG<br /> **** ***** ********** **************************************<br /> AJ415520 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Y16238 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Y16232 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Prorocentrum sp. TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> AJ415519 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Y16239 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Y16233 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Y16237 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTG<br /> Y16234 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACTTG<br /> Y16235 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACTTG<br /> Y16236 TTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACTTG<br /> ********************************************************* **<br /> AJ415520 CTAAATAGTTACACGTAACTTCGGTTACGTGGGCAACTTCTTAGAGGGACTTTG-CGTGT<br /> Y16238 CTAAATAGTTACACGTAACTTCGGTTACGTGGGCAACTTCTTAGAGGGACTTTG-CGTGT<br /> Y16232 CTAAATAGTTACACGTAACTTCGGTTACGTGGGCAACTTCTTAGAGGGACTTTG-CGTGT<br /> Prorocentrum sp. CTAAATAGTTACACGTAACTTCGGTTACGTGGGCAACTTCTTAGAGGGACTTTG-CGTGT<br /> AJ415519 CTAAATAGTTACACGTAACTCCGGTTACGTGGGCAACTTCTTAGAGGGACTTTG-CGTGT<br /> Y16239 CTAAATAGCTACACATAACTCCAGTCATGTGGGCAGCTTCTTAGAGGGACTTTG-TGTGT<br /> Y16233 CTAAATAGTTACATGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTG-CGTGT<br /> Y16237 CTAAATAGTTACACGTAACTCTGGCTACGTGGGCAGCTTCTTAGAGGGACTTTGCTGTGT<br /> Y16234 CTGAATAGCTACATCTAACTCCGGTTACATGGGCAGCTTCTTAGAGGGACTTTGCTGTGT<br /> Y16235 CTGAATAGCTACATCTAACTCCGGTTACATGGGCAGCTTCTTAGAGGGACTTTGCTGTGT<br /> Y16236 CTGAATAGCTACATCTAACTCCGGTTACATGGGCAGCTTCTTAGAGGGACTTTGCTGTGT<br /> ** ***** **** *** * * * ****** ****************** ****<br /> AJ415520 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16238 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16232 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Prorocentrum sp. CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> AJ415519 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16239 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16233 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16237 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16234 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16235 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> Y16236 CTAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC<br /> ******************************************<br /> <br /> H×nh 6. So s¸nh tr×nh tù nucleotit cña ®o¹n gien 18S rDNA cña Prorocentrum sp. víi 10 tr×nh tù cña ®o¹n<br /> gien nµy cña 9 loµi Prorocentrum kh¸c: P. minimum cã md sè t¹i Ng©n gµng gien quèc tÕ lµ AJ 415520 vµ<br /> Y16238, P. mexicanum-Y16232; P. micans-AJ415519; P. emarginatum-Y16239; P. panamensis-Y16233;<br /> P. concavum-Y16237; P. arenarium-Y16234; P. lima-Y16235; P. maculosum-Y16236.<br /> AF208245 GCACGCATCCAATAGATTCACTGTGAACTCGA-ACTCGTGAGGGTCTGGGTTGGGGTGGA<br /> Prorocentrum sp. GCACGCATCCAATCGATTCACTGTGAACGAAC-TTTTGTGAGGGTCTGGGTGAGGGTGGA<br /> AF208244 GCACGCATCCATTCGAATCATTGTGAACAACA-GTTGGTGAGGCTCTGGGTGGGGATGGA<br /> AF352371 GCACGCATCCATTCGAATCATTGTGAACAACA-GTTGGTGAGGCTCTGGGTGGGGATGGA<br /> AF352370 GCACGCATCCATTCGAATCATTGTGAACAACA-GTTGGTGAGGCTCTGGGTGGGGATGGA<br /> AF208246 GCACGCATCCAACCAAATCACCGTGAATAACACGTTGGTGAAGCTCTGGGTGGGGATGGA<br /> *********** * *** ***** * **** * ******* ** ****<br /> AF208245 GATAGCATCAATGCCCTTATGCAGGCGCTCGAGGGCAGTAAGCCAGGATTGGACTGTCTT<br /> Prorocentrum sp. GATTGCATCAATCCCCTTATGCAGGCGCTCGAGGGCAGTAAGCCAGGCTTGGATTGTCTT<br /> 87<br />  AF208244 GATAGCATCGATGCCCCCATGCAGAGACTCAAGGGCAGCAAGCCAGGCTCAGACCGTCTT<br /> AF352371 GATAGCATCGACGCCCCCATGCAGAGACTCAAGGGCAGCAAGCCAGGCTCAGACCGTCTT<br /> AF352370 GATAGCATCGACGCCCCCATGCAGAGACTCAAGGGCAGCAAGCCAGGCTCAGACCGTCTT<br /> AF208246 GATAGCATCGATTCCCCCATGCAGAC-TTCAAGGGCACTGGGCCAGGTGCGGACCGTCTT<br /> *** ***** * *** ****** ** ****** ****** ** *****<br /> AF208245 CCTTTCCTGTCCCTGCTGCCATGTCTTCAACTTGCTACAAAGCATTTATTGTTCCATTTG<br /> Prorocentrum sp. CCTTGCCTGCCCCTGCTGCCATCGCTTCAACTTGCTATTCAGCATTTATTGTTTC--TTT<br /> AF208244 CTGTGCCTGTCCTTGCTGTCG-GGTGTCTTCCTGATCTTCTGTGTTTTTGAATTC----T<br /> AF352371 CTGTGCCTGTCCTTGCTGTCG-GGTGTCTTCCTGATCTTCTGTGTTTTTGAATTC----T<br /> AF352370 CTGTGCCTGTCCTTGCTGTCG-GGTGTCTTCCTGATCTTCTGTGTTTTTGAATTC----T<br /> AF208246 CTGCATCTGCGCCTGCTGCCA-ATTGTCTTTTGACTTATTGGTTTCTTCATATCC----T<br /> * *** * ***** * ** * * * * * *<br /> AF208245 TTCTCGAGTGGTTATCCACTTGTTTATTGTATTACAACTTTCAGCGACGGATGTCTCGGC<br /> Prorocentrum sp. TCTTCGAGTGGTTATCCACTTATTCATCGCATTACAACTTTCAGCGACGGATGTCTCGGC<br /> AF208244 CTCCTGAGTGGTC-TCCACTCTCACATCTACTTACAACTTTCAGCGACGGATGTCTCGGC<br /> AF352371 CTCCTGAGTGGTC-TCCACTCTCACATCTACTTACAACTTTCAGCGACGGATGTCTCGGC<br /> AF352370 CTCTTGAGTGGTC-TCCACTCTCACATCTACTTACAACTTTCAGCGACGGATGTCTCGGC<br /> AF208246 ATCTCTTGTGGCTTGTTCCACATGTCTTCTCATACAACTTTCAGCGATGGATGTCTCGGC<br /> **** * * *************** ************<br /> AF208245 TCGAACAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAATTCCGT<br /> Prorocentrum sp. TCGAACAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAATTCCGT<br /> AF208244 TCGAACAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAATTCCGT<br /> AF352371 TCGAACAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAATTCCGT<br /> AF352370 TCGAACAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAATTCCGT<br /> AF208246 TCGAACAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAATTCCGT<br /> ************************************************************<br /> AF208245 GAACCAATAGGGACTTGAACGTATACTGCGCTTTCGGGATATCCCTGAAAGCATGCCTGC<br /> Prorocentrum sp. GAACCAATAGGGACTTGAACGTACACTGCGCTTTCGGGATATCCCTGAAAGCATGCCTGC<br /> AF208244 GAACCAATAGGGACTTGAACGTATACTGCGCTTTCGGGATATCCCTGAAAGCATGCCTGC<br /> AF352371 GAACCAATAGAGACTTGAACGTATACTGCGCTTTCGGGATATCCCTGAAAGCATGCCTGC<br /> AF352370 GAACCAATAGGGACTTGAACGTATACTGCGCTTTCGGGATATCCCTGAAAGCATGCCTGC<br /> AF208246 GAACCAATAGGGACTTGAACGTATACTGCGCTTTCGGGATATCCCTGAAAGCATGCCTGC<br /> ********** ************ ************************************<br /> AF208245 TTCAGTGTCTATTCTTTTTCATTCCAGCGATCTTGGGTTTCCAGCGTCGCTTGTGTGTCT<br /> Prorocentrum sp. TTCAGTGTCTATTCTTTTTCATTCCAGCGACCTGGTTTCTCCAGAGTCGCTTGCGTGTCT<br /> AF208244 TTCAGTGTCTATTCTGTATCATTCCAGCT-TCTGGCCTGTCCAGAA-CGCTTGTGTGTTT<br /> AF352371 TTCAGTGTCTATTCTGTATCATTCCAGCT-TCTGGCCTGTCCAGAA-CGCTTGTGTGTTT<br /> AF352370 TTCAGTGTCTATTCTGTATCATTCCAGCT-TCTGGCATGTCCAGAA-CGCTTGTGTGTTT<br /> AF208246 TTCAGTGTCTAATTCATTTCATTCCAGCAACCTGGT-TTTCCAGTGCTGCTTGGGTGTAT<br /> *********** * * ********** ** * * ***** ***** **** *<br /> AF208245 TTGTGCGTTAGAGCGCTCGCCTTGCGCAGCCTTTGACGCATTTAATGCA-CAGGGACCCC<br /> Prorocentrum sp. TTGTGCGTTAGGGCGCTCGCTTCTTGCGGCCCTTGACGCATTCAATGCA-CAGGGACCCC<br /> AF208244 CTGTGTGCCAGGGCGCCC------TGCGGCCTCTGGCGCATTCAGTGCA-CAGGGTCTTC<br /> AF352371 CTGTGTGCCAGGGCGCCC------TGCGGCCTCTGGCGCATTCAGTGCA-CAGGGTCTTC<br /> AF352370 CTGTGTGCCAGGGCGCCC------TGCGGCCTCTGGCGCATTCAGTGCA-CAGGGTCTTC<br /> AF208246 TTGTGTGTCAGTGTGCTT-------TTTGCCTTTGACACATTGAGCTCATCAGGTTTTCC<br /> **** * ** * ** *** ** * **** * ** **** *<br /> AF208245 TCGCACAAGCAACTAGAAGAGCCTC--TTGGCGTTTCCTTGTTGTCTTGCCTGCGAGGAG<br /> Prorocentrum sp. TTGCACAAGCAACTAGAAGAGCCTC--TGGGCGTTTCCTTGTTGTCTTGCATGCGAGCGG<br /> AF208244 CCACGCAAGCAACTAGAAGAGTGTC--TCTGATGCTATCTGTTGCCTTGTTGTTGGGCAT<br /> AF352371 CCACGCAAGCAACTAGAAGAGTGTC--TCTGATGCTATCTGTTGCCTTGTTGTTGGGTAT<br /> AF352370 CCACGCAAGCAACTAGAAGAGTGTC--TCTGATGCTATCTGTTGCCTTGTCGTTGGGCAT<br /> AF208246 TTGCGCAAGCAATTAGAAGGGTGTTATTATGACGCTATCTGTTTGCTTGTTTGCTAAGGG<br /> * ******* ****** * * * * * **** ****<br /> AF208245 GGCCTTGCCATCCAGTGCCTAGCGCACTCCA<br /> Prorocentrum sp. GGCTTTGCCATCCAGTGCAGAACGCACTCCA<br /> AF208244 GGCCTTGCTGTCTAGTGCCCAGCGCACTCCT<br /> AF352371 GGCCTTGCTGTCTAGTGCCCAGCGCACTCCT<br /> AF352370 GGCCTTGCTGTCTAGTGCCCAGCGCACTCCT<br /> AF208246 AAGCTTGGCTTGCAGTGCCTTGTGCACTCAA<br /> *** * ***** ******<br /> <br /> H×nh 7. So s¸nh tr×nh tù nucleotit cña ®o¹n gien ITS1-5,8S-ITS2 cña Prorocentrum sp. víi 5 tr×nh<br /> tù nucleotit cña ®o¹n gien nµy cña 3 loµi kh¸c: P. micans víi md sè ký hiÖu t¹i Ng©n hµng gien<br /> quèc tÕ lµ AF208245, P. minimum-AF208244, AF352371, AF352370 vµ P. triestinum-AF208246.<br /> 88<br />  B¶ng 2<br /> Tû lÖ % t−¬ng ®ång (ma trËn tam gi¸c trªn) vµ kho¶ng c¸ch di truyÒn (ma trËn tam gi¸c d−íi)<br /> cña ®o¹n gien 18S rDNA gi÷a c¸c loµi trong chi Prorocentrum<br /> Tû lÖ % t−¬ng ®ång<br /> 1 2 3 4 5 6 7 8 9 10 11<br /> 1 99,6 99,8 99,9 96,4 94,3 94,0 94,3 96,7 99,6 95,7 1 AJ415519<br /> 2 0,4 99,6 99,7 96,4 94,2 93,9 99,2 96,6 100 95,6 2 AJ415520<br /> Kho¶ng c¸ch di truyÒn<br /> <br /> <br /> <br /> <br /> 3 0,2 0,4 99,9 96,4 94,1 93,8 94,1 96,5 99,6 95,6 3 Prorocentrum sp.<br /> 4 0,1 0,3 0,1 96,4 94,2 94,0 94,2 96,5 99,7 95,7 4 Y16232<br /> 5 3,7 3,7 3,7 3,6 92,9 92,9 92,7 94,2 96,3 92,8 5 Y16233<br /> 6 5,8 5,9 6,1 5,9 7,3 99,9 99,0 94,3 94,2 91,8 6 Y16234<br /> 7 6,0 6,1 6,2 6,1 7,2 0,1 98,5 93,9 93,8 91,8 7 Y16235<br /> 8 5,9 5,9 6,1 6,0 7,5 1,0 1,1 94,2 94,2 92,0 8 Y16236<br /> 9 3,3 3,4 3,5 3,4 6,0 5,9 6,1 5,9 96,6 93,6 9 Y16237<br /> 10 0,4 0,0 0,4 0,3 3,7 5,9 6,1 5,9 3,4 95,6 10 Y16238<br /> 11 4,4 4,6 4,6 4,5 7,6 8,9 8,8 8,7 6,9 4,6 11 Y16239<br /> 1 2 3 4 5 6 7 8 9 10 11<br /> B¶ng 3<br /> Tû lÖ % t−¬ng ®ång (ma trËn tam gi¸c trªn) vµ kho¶ng c¸ch di truyÒn (ma trËn<br /> tam gi¸c d−íi) cña ®o¹n gien ITS1-5,8S-ITS2 gi÷a c¸c loµi trong chi Prorocentrum<br /> Tû lÖ % t−¬ng ®ång<br /> 1 2 3 4 5 6<br /> 1 80,3 79,6 99,3 99,5 79,9 1 AF208244<br /> Kho¶ng c¸ch<br /> <br /> <br /> <br /> <br /> 2 25,2 77,4 80,1 79,6 91,5 2 AF208245<br /> di ruyÒn<br /> <br /> <br /> <br /> <br /> 3 28,2 30,9 79,2 79,0 77,4 3 AF208246<br /> 4 0,7 24,9 28,5 99,1 79,9 4 AF352370<br /> 5 0,5 25,7 28,8 0,9 79,1 5 AF352371<br /> 6 24,5 10,3 32,5 24,5 25,4 6 Prorocentrum sp.<br /> 1 2 3 4 5 6<br /> <br /> 6. X©y dùng c©y ph¸t sinh chñng lo¹i cña Prorocentrum sp. n»m ngay c¹nh loµi P.<br /> mét sè loµi Prorocentrum mexicanum trong c©y ph¸t sinh chñng lo¹i, cã hÖ<br /> Dùa vµo kho¶ng c¸ch di truyÒn cña ®o¹n gien sè ®ång d¹ng di truyÒn cao nhÊt 99,9% và chóng<br /> 18S rDNA tõ mét sè loµi Prorocentrum kh¸c nhau chØ cã kh¸c biÖt nhau ë 1 nucleotit khi so s¸nh<br /> vµ b»ng ch−¬ng tr×nh DNASTAR ®Ó tÝnh to¸n kh¶ tr×nh tù nucleotit cña ®o¹n gien 18S rDNA. Sù<br /> n¨ng lín nhÊt cã thÓ x¶y ra, chóng t«i x©y dùng kh¸c biÖt ë 1 nucleotit nµy cã thÓ chØ lµ sù kh¸c<br /> c©y ph¸t sinh chñng lo¹i cña c¸c loµi biÖt ë trong cïng loµi P. mexicanum. Nh− vËy,<br /> Prorocentrum nµy (h×nh 8). C©y ph¸t sinh chñng Prorocentrum sp. cã thÓ lµ loµi Prorocentrum<br /> lo¹i cho thÊy c¸c loµi nµy ®−îc chia thµnh 2 nhãm: mexicanum và kÕt qu¶ ph©n lo¹i nµy còng phï hîp<br /> nhãm thø nhÊt cã duy nhÊt 1 loµi lµ P. víi viÖc ph©n lo¹i dùa vµo c¸c ®Æc ®iÓm h×nh th¸i<br /> emarginatum vµ nhãm thø 2 gåm c¸c loµi cßn l¹i; mµ chóng t«i ®d ®Ò cËp ë trªn.<br /> trong ®ã, l¹i ®−îc chia ra lµm 2 nhãm nhá: nhãm Sau khi ®d x¸c ®Þnh ®−îc Prorocentrum sp. lµ<br /> nhá thø nhÊt gåm c¸c loµi: P. aenarium, P. lima, loµi Prorocentrum mexicanum, tr×nh tù nucleotit cña<br /> P. maculosum, P. concavum vµ P. panamensis vµ ®o¹n gien ITS1-5,8S-ITS2 cña loµi Prorocentrum<br /> nhãm nhá thø 2 gåm: Prorocentrum sp., P. mexicanum nµy ®d ®−îc ®¨ng ký t¹i Ng©n hµng gien<br /> mexicanum, P. micans vµ P. minimum. quèc tÕ, víi sè ®¨ng ký ®−îc cÊp lµ AY 886 763.<br /> 89<br />  Y16234 P. arenarium<br /> Y16235 P. lima<br /> Y16236 P. maculosum<br /> Y16237 P. concavum<br /> Y16233 P. panamensis<br /> Prorocentrum sp.<br /> Y16232 P. mexicanum<br /> AJ415519 P. micans<br /> AJ415520 P. minimum<br /> Y16238 P. minimum<br /> Y16239 P. emarginatum<br /> 3,9<br /> 2 0<br /> <br /> H×nh 8. C©y ph¸t sinh chñng lo¹i cña mét sè loµi Prorocentrum dùa trªn so s¸nh<br /> tr×nh tù nucleotit cña ®o¹n gien 18S rDNA.<br /> <br /> <br /> III. KÕt luËn 4. §Æng DiÔm Hång vµ cs., 2004: TuyÓn tËp<br /> b¸o c¸o khoa häc Héi nghÞ khoa häc “BiÓn<br /> 1. Chóng t«i ®d ph©n lËp, t¸ch dßng vµ ®äc §«ng 2002”: 424-436. Nha Trang.<br /> thµnh c«ng tr×nh tù nucleotit cña c¸c ®o¹n gien 18S<br /> rDNA và ITS1-5,8S-ITS2 cña loài Prorocentrum 5. §Æng DiÔm Hång vµ cs., 2002: T¹p chÝ<br /> sp. ph©n lËp ®−îc t¹i H¶i Phßng. Khoa häc và C«ng nghÖ, 40: 161-167.<br /> 2. Dùa trªn c¸c ®Æc ®iÓm h×nh th¸i vµ so 6. §Æng §×nh Kim, §Æng Hoàng Ph−íc<br /> s¸nh tr×nh tù nucleotit cña c¸c ®o¹n gien 18S HiÒn, 1999: C«ng nghÖ sinh häc Vi t¶o.<br /> rDNA vµ ITS1-5,8S-ITS2 cña loµi Prorocentrum Nxb. N«ng nghiÖp, Hà Néi.<br /> sp. víi c¸c tr×nh tù t−¬ng øng cña c¸c 7. Faust M. A., 1997: J. Phycol., 33: 851-858.<br /> Prorocentrum kh¸c ®d ®−îc c«ng bè t¹i Ng©n<br /> 8. NguyÔn §øc B¸ch vµ cs., 2003: T¹p chÝ<br /> hµng gien quèc tÕ, ®d cho phÐp chóng t«i x¸c<br /> Sinh häc, 25(3): 1-5.<br /> ®Þnh loµi t¶o nµy lµ Prorocentrum mexicanum.<br /> 9. Taylor F. J. R., 1993: The species problem<br /> 3. Tr×nh tù nucleotit cña ®o¹n gien ITS1-<br /> and its impact on harmful phytoplankton<br /> 5,8S-ITS2 cña loµi Prorocentrum mexicanum<br /> studies, with emphasis on dinoflagellate<br /> thu ®−îc t¹i H¶i Phßng cã sè ®¨ng ký t¹i Ng©n morphology. In: T. J. Smayda and Y.<br /> hµng gien quèc tÕ lµ AY 886 763. Shimizu (eds), Toxic Phytoplankton Blooms<br /> in the Sea: 81-86. New York, Elsevier<br /> Tµi liÖu tham kh¶o Science Inc.<br /> 1. Altamirano R. C. and Beltran A. P. S., 10. Takano Y. and Horiguchi T., 2004:<br /> 2003: J. Phycol., 39: 221-225. Phycological Research, 52: 107-116.<br /> 2. Asai R. et al., 2003: Phycological Research, 11. Witek B., Plinski M., 2000: Oceanologia,<br /> 51: 118-125. 41(2): 29-36.<br /> 3. Casas M. S. et al., 2002: Diseases of 12. Grzebyk D. et al., 1998: J. Phycol., 34:<br /> Aquatic Organisms, 50: 51-65. 1055-1068.<br /> <br /> <br /> <br /> 90<br />  TAXONOMY OF PROROCENTRUM SP. SAMPLED<br /> FROM HAIPHONG CITY BY BASING ON THE NUCLEOTIDE<br /> SEQUENCES OF THE 18S rDNA And ITS1-5.8S-ITS2 GENE FRAGMENTS<br /> <br /> Dang Diem Hong, Hoang Minh Hien, Hoang Lan Anh, Chu Van Thuoc<br /> <br /> <br /> Summary<br /> <br /> The nucleotide sequences of the 18S rDNA and ITS1-5.8S-ITS2 gene fragments were determined in<br /> Prorocentrum sp., which was collected from Haiphong city, Vietnam in 2004. The length of the 18S rDNA<br /> and ITS1-5.8S-ITS2 gene fragments in Prorocentrum sp. were 1059 bp and 657bp, respectly. By comparing<br /> the Prorocentrum sp. sequence data with the published sequences of P. lima, P. arenarium, P. maculosum, P.<br /> concavum, P. panamensis, P. micans, P. minimum, P. mexicanum, P. emarginatum and P.triestinum in the<br /> GeneBank and constructing the phylogenetic tree, the phylogenetic relationships between Prorocentrum sp.<br /> and the ten other Prorocentrum species were established. The classification of Prorocentrum species based on<br /> the 18S rRNA gene coincided with the classification based on the ITS1-5.8S-ITS2 gene. The results showed<br /> that the genetic distances between the species within these two groups were low. The similar of these<br /> Prorocentrum species was 94% and they were divided into two groups. The first group included P.<br /> emarginatum. The second group included other species with 2 subgroups; the first subgroup included P.<br /> arenarium, P. lima, P. maculosum, P. concavum and P. panamensis and the second subgroup included<br /> Prorocentrum sp., P. mexicanum, P. micans and P. minimum. Among these species, Prorocentrum sp. had a<br /> close phylogenetic relationship with P. mexicanum (99.9%). The morphological characteristics of<br /> Prorocentrum sp. also showed a relatively high similarity with P. mexicanum. It has been shown that<br /> Prorocentrum sp. collected from Haiphong belonged to the species P. mexicanum. The nucleotide sequence of<br /> the ITS1-5.8S-ITS2 gene fragments of P. mexicanum (collected from Haiphong) was submitted to the<br /> GeneBank with accession number-AY886 763.<br /> <br /> Ngµy nhËn bµi: 11-05-2005<br /> <br /> <br /> <br /> <br /> 91<br />