phân lập và tuyển chọn vi khuẩn sinh bacteriocin từ động vật thân mềm hai vỏ sống ở biển

B GIÁO D C VÀ ÀO T O TRƯ NG H I H C NHA TRANG *** TH H NG NHI PHÂN L P VÀ TUY N CH N VI KHU N SINH BACTERIOCIN T NG V T THÂN M M HAI V S NG BI N LU N VĂN TH C SĨ Nha Trang - 2013 B GIÁO D C VÀ ÀO T O TRƯ NG H I H C NHA TRANG *** TH H NG NHI PHÂN L P VÀ TUY N CH N VI KHU N SINH BACTERIOCIN T NG V T THÂN M M HAI V S NG BI N Chuyên ngành: Nuôi tr ng th y s n Mã s : 60 62 03 01 LU N VĂN TH C SĨ NGƯ I HƯ NG D N KHOA H C : TS NGUY N VĂN DUY Nha Trang - 2013 i L i cam oan Tôi xin cam oan k t qu c a lu n văn “Phân l p n ch n vi khu n sinh bacteriocin t ng v t thân m m hai v s ng ch n nghiên c u bi n” thu c tài “Phân l p, n c i m sinh h c c a vi khu n bi n sinh bacteriocin dùng làm thu c a nuôi tr ng th y s n” (Mã s : 106.03 – 2011.34) Qu phát tri n khoa h c công ngh Qu c gia (NAFOSTED) tài tr - Trư ng i h c Nha Trang ch trì - TS Nguy n Văn Duy làm ch nhi m, c th c hi n t tháng 02 năm 2012 n tháng 03 năm 2013 xác Các s li u, k t qu trình bày lu n văn hồn tồn trung th c chưa c công b b t c cơng trình khoa h c khác t i th i i m Các thơng tin trích d n lu n văn u ã c ch rõ ngu n g c Tác gi lu n văn H TH H NG NHI ii L i c m ơn Tơi xin bày t lịng bi t ơn sâu s c s kính tr ng t i th y hư ng d n TS Nguy n Văn Duy – Vi n Công ngh Sinh h c Môi trư ng - Trư ng Trang, ã nh hư ng t o m i i u ki n i h c Nha tơi hồn thành lu n văn Xin chân thành cám ơn Ban Giám hi u nhà trư ng, th y cô Khoa Nuôi tr ng th y s n - Trư ng i h c Nha Trang ã t o i u ki n giúp th i gian tham gia khóa h c Xin chân thành c m ơn Lãnh h c Mơi trư ng ã nhi t tình giúp o Vi n, cán b thu c Vi n Công ngh Sinh th i gian th c hi n lu n văn Xin c m ơn Qu phát tri n khoa h c công ngh Qu c gia (NAFOSTED) ã h tr kinh phí thu c tài “Phân l p, n ch n nghiên c u c i m sinh h c c a vi khu n bi n sinh bacteriocin dùng làm thu c a nuôi tr ng th y s n” (Mã s : 106.03 – 2011.34) giúp th c hi n lu n văn t t nghi p L i c m ơn sâu s c t i gia ình, b n bè ã ln bên c nh giúp su t th i gian h c t p th c hi n M t l n n a, xin c m ơn t t c s giúp , ng viên tài quý báu ó Tác gi lu n văn H Th H ng Nhi iii M CL C L i cam oan i L i c m ơn ii DANH M C CÁC CH VI T T T v DANH M C B NG vi DANH M C HÌNH vii M U CHƯƠNG T NG QUAN TÀI LI U 1.1 T ng quan v bacteriocin 1.1.1 Khái ni m v bacteriocin 1.1.2 Phân lo i bacteriocin 1.1.2.1 Bacteriocin c a vi khu n 1.1.2.2 Bacteriocin t sinh v t c (Archaea) 1.1.3 M t s tính ch t hóa lý c a bacteriocin 1.1.3.1 b n enzyme 1.1.3.2 b n nhi t 10 1.1.3.3 b n pH 10 1.1.4 Cơ ch ho t 1.1.5 ng c a bacteriocin 10 ng d ng c a bacteriocin 12 1.1.5.1 B o qu n th c ph m 12 1.1.5.2 B o v s c kh e ngư i v t nuôi c n 12 1.2 Tri n v ng s d ng vi khu n sinh bacteriocin làm thu c a nuôi tr ng th y s n 14 1.2.1 Tình hình d ch b nh nuôi tr ng th y s n 14 1.2.2 Nh ng gi i pháp m i phòng tr d ch b nh nuôi tr ng th y s n b n v ng 15 1.2.3 Tri n v ng s d ng vi khu n sinh bacteriocin dùng làm thu c a nuôi tr ng th y s n 17 1.3 Tình hình nghiên c u bacteriocin t vi khu n bi n 20 1.3.1 Trên th gi i 20 1.3.2 T i Vi t Nam 25 CHƯƠNG V T LI U VÀ PHƯƠNG PHÁP NGHIÊN C U 27 2.1 i tư ng, th i gian a i m nghiên c u 27 iv 2.2 Nguyên v t li u 27 2.2.1 M u tu hài 27 2.2.2 Ch ng vi khu n ch th 27 2.2.3 Hóa ch t, mơi trư ng thu c th 27 2.2.4 Thi t b chuyên d ng 29 2.3 Phương pháp nghiên c u 30 2.3.1 Phân l p vi khu n 30 2.3.2 Nuôi c y b o qu n ch ng vi khu n 30 2.3.3 Xác nh ho t tính sinh bacteriocin 31 2.3.3.1 Xác nh ho t tính kháng khu n 31 2.3.3.2 Xác nh b n ch t c a ch t kháng khu n 32 2.3.4 Xác nh m t s tính ch t c a d ch bacteriocin thơ 32 2.3.4.1 Th b n v i enzyme c a d ch bacteriocin thô 32 2.3.4.2 Xác nh b n v i nhi t c a d ch bacteriocin thô 32 2.3.4.3 Xác nh b n v i pH c a d ch bacteriocin thô 33 2.3.5 Xác nh hình thái khu n l c t bào vi khu n 33 2.3.5.1 Xác nh hình thái khu n l c 33 2.3.5.2 Nhu m Gram t bào vi khu n 33 2.3.6 nh danh vi khu n b ng phương pháp gi i trình t o n gen 16S rDNA rpoB 34 2.3.7 Phân tích quan h phát sinh lồi 35 2.3.8 X lý th ng kê 35 CHƯƠNG K T QU NGHIÊN C U VÀ TH O LU N 37 3.1 Phân l p ch ng vi khu n t th t tu hài 37 3.2 Tuy n ch n ch ng vi khu n có ho t tính kháng khu n 37 3.3 Xác nh b n ch t bacteriocin c a ch t kháng khu n 41 3.4 nh hư ng c a enzyme, nhi t pH n ho t tính kháng khu n c a ch ng n ch n 45 3.5 c i m hình thái khu n l c c a ch ng nghiên c u 50 3.6 nh danh ch ng vi khu n sinh bacteriocin d a trình t o n gen 16S rDNA rpoB 51 K T LU N VÀ KI N NGH 58 TÀI LI U THAM KH O 59 v DANH M C CÁC CH STT Ký hi u VI T T T Ch vi t t t ATP Adenosin triphosphat BLIS Bacteriocin-like substance (ch t tương t bacteriocin) CFU Colony Forming Unit ( ơn v hình thành khu n l c) DNA Deoxyribonucleotide Acid FAO FDA GRAS NTTS Nuôi tr ng th y s n OD Optical Density (M t 10 TCBS Thiosulfate Citrate Bile Salts Sucrose 11 TSA Tryptone Soya Agar 12 TSB Tryptone Soya Broth Food and Agriculture Organization (T ch c LHQ v lương th c nông nghi p) Food and Drug Administration (C c qu n lý th c ph m dư c ph m Hoa Kỳ) Generally recognized as safe ( c cơng nh n r ng rãi an tồn) quang) vi DANH M C B NG B ng 1.1 So sánh gi a bacteriocin kháng sinh B ng 1.2 Bacteriocin c a vi khu n sinh v t c B ng 1.3 Tính ch t hóa lý c a m t s bacteriocin t vi khu n .9 B ng 1.4 Các ch ng vi khu n bi n sinh bacteriocin 22 B ng 1.5 Bacteriocin t vi khu n lactic c phân l p t ng v t bi n 24 B ng 2.1 Thu m u Tu hài phân l p vi khu n bi n .27 B ng 3.1 S ch ng vi khu n l n phân l p 37 B ng 3.2 Ho t tính kháng khu n c a c a ch ng phân l p i v i ba ch ng vi khu n ích 38 B ng 3.3 nh hư ng c a trypsin proteinase K n ho t tính kháng khu n c a d ch bacteriocin thô c a ch ng n ch n 44 B ng 3.4 nh hư ng c a m t s enzyme th y phân lên ho t tính kháng khu n c a d ch bacteriocin thô t ch ng n ch n i v i vi khu n ch th Entercoccus faecalis B1.1 45 B ng 3.5 b n nhi t c a d ch bacteriocin thô t ch ng n ch n 46 B ng 3.6 nh hư ng c a pH n ho t tính kháng khu n c a d ch bacteriocin thô t ch ng n ch n 48 B ng 3.7 c i m khu n l c t bào c a ch ng vi khu n sinh bacteriocin 51 B ng 3.8 So sánh trình t H108 v i trình t tương B ng 3.9 So sánh trình t trình t tương o n gen 16S rDNA c a ch ng H77 (1064 nucleotide) v i o n gen rpoB c a ch ng H108 (857 nuleotide) v i ng Genbank b ng công c BLAST .56 B ng 3.11 So sánh trình t trình t tương ng Genbank b ng công c BLAST .53 ng Genbank b ng công c BLAST .55 B ng 3.10 So sánh trình t trình t tương o n gen 16S rDNA c a ch ng H9, H18, H51, H61 o n gen rpoB c a ch ng H77 (904 nucleotide) v i ng Genbank b ng công c BLAST .56 vii DANH M C HÌNH Hình 1.1 C u trúc c a m t s bacteriocin Hình 1.2 Cơ ch ho t c trưng ng c a bacteriocin 11 Hình 1.3 Quy trình n ch n m t probiotic ni tr ng th y s n 19 Hình 1.4 S lư ng báo nghiên c u v bacteriocin m i chu kỳ 10 năm t 1949-2010 20 Hình 2.1 Sơ n i dung nghiên c u c a lu n văn 36 Hình 3.1 Khu n l c ĩa th ch pha loãng 10-6 sau 24 gi ni c y .37 Hình 3.2 T l ch ng vi khu n có ho t tính kháng v i vi khu n ch th ba l n phân l p 39 Hình 3.3 Vịng kháng khu n c a ch ng vi khu n phân l p i v i ch ng ch th Vibrio alginolyticus V3.3 môi trư ng TCBS 40 Hình 3.4 Vịng kháng khu n c a ch ng vi khu n phân l p i v i ch ng ch th Entercoccus faecalis B1.1 môi trư ng TSA 40 Hình 3.5 Vịng kháng khu n c a ch ng vi khu n phân l p i v i ch ng ch th V parahaemolyticus C1 môi trư ng TCBS 41 Hình 3.6 Vịng kháng khu n c a d ch bacteriocin c a ch ng H18 H76 sau x lý v i proteinase K trypsin, vi khu n ch th Entercoccus faecalis B1.1 42 Hình 3.7 Vịng kháng khu n c a d ch bacteriocin c a hai ch ng H18, X1.10 sau x lý v i trypsin proteinase K, vi khu n ch th V alginolyticus V3.3 (A) V parahaemolyticus C1 (B) 43 Hình 3.8 nh hư ng c a lipase α-amylase ho t tính kháng khu n c a d ch bacteriocin thô t ch ng n ch n .47 Hình 3.9 nh hư ng c a nhi t i v i ho t tính c a d ch bacteriocin thơ c a ch ng H108 H18 47 Hình 3.10 nh hư ng c a pH Hình 3.11 n ho t tính c a d ch bacteriocin thơ t ch ng H108 49 c i m hình thái khu n l c c a ch ng H77 H108 49 Hình 3.12 Hình nh nhu m Gram c a ch ng sinh bacteriocin nghiên c u 50 viii Hình 3.13 S n ph m 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asociated Vibrio spp", Packistan Journal of Pharmaceutical Sciences, 22, pp 162-167 105 Zhang, X.H and Austin, B (2000), "Pathogenicity of Vibrio harveyi to salmonids", Journal of Fish Diseases, 23, pp 93-102 PH L C PL Thành ph n ph n ng PCR Thành ph n Th tích (µl) Buffer Tag 10X MgCl2 (25 mM) 10 dNTP (10 mM) 10 Primer xuôi (16S-27F) Primer ngư c (16S-1492R) Taq polymerase 0,2 DNA H2 O 23,8 T ng th tích 50 Chu trình nhi t th c hi n ph n ng PCR cho khu ch i o n gen 16S rDNA 94oC - phút 94oC – phút 62oC – 1phút 72oC - phút 30 giây 72oC - phút 4oC - ∞ 30 chu kỳ PL Thành ph n ph n ng PCR Thành ph n Th tích (µl) Buffer Tag 10X MgCl2 (25 mM) 10 dNTP (10 mM) 10 Primer xuôi (16S-27F) Primer ngư c (16S-1492R) Taq polymerase 0,2 DNA H2 O 23,8 T ng th tích Chu trình nhi t th c hi n ph n 50 ng PCR cho khu ch i o n gen rpoB c a ch ng H108 94oC - phút 94oC – 30 giây 52oC – 30 giây 30 chu kỳ 72oC - phút 20 giây 72oC - phút 44 C - ∞ Chu trình nhi t th c hi n ph n ng PCR cho khu ch i o n gen rpoB c a ch ng H77 94oC - phút 94oC – 30 giây 50oC – 30 giây 72oC - phút 20 giây 72oC - phút 44 C - ∞ 30 chu kỳ PL 3: Trình t PL 3.1 Trình t o n gen 16S rDNA rpoB c a ch ng vi khu n phân l p o n gen 16S rDNA c a ch ng H9 (KC894665) TACGGGAGGC AGCAGTGGGG AATATTGCAC AATGGGCGCA AGCCTGATGC 50 51 AGCCATGCCG CGTGTATGAA GAAGGCCTTC GGGTTGTAAA GTACTTTCAG 100 101 CGGGGAGGAA GGTGTTGTGG TTAATAACCA CAGCAATTGA CGTTACCCGC 150 151 AGAAGAAGCA CCGGCTAACT CCGTGCCAGC AGCCGCGGTA ATACGGAGGG 200 201 TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG 250 251 TTAAGTCAGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTTGAAA 300 301 CTGGCAGGCT TGAGTCTCGT AGAGGGGGGT AGAATTCCAG GTGTAGCGGT 350 351 GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG 400 401 ACGAAGACTG ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA 450 451 TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG GTTGTGCCCT 500 501 TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA 550 551 CGGCCGCAAG GTTAAAACTC AAATGAATTG ACGGGGGCCC GCACAAGCGG 600 601 TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGTCTT 650 651 GACATCCAGA GAATCCTGCA GAGATGCGGG AGTGCCTTCG GGAACTCTGA 700 701 GACAGGTGCT GCATGGCTGT CGTCAGCTCG TGTTGTGAAA TGTTGGGTTA 750 751 AGTCCCGCAA CGAGCGCAAC CCTTATCCTT TGTTGCCAGC GGTCCGGCCG 800 801 GGAACTCAAA GGAGACTGCC GGTGATAAAC CGGAGGAAGG TGGGGATGAC 850 851 GTCAAGTCAT CATGGCCCTT ACGACCAGGG CTACACACGT GCTACAATGG 900 901 CGCATACAAA GAGAAGCGAC CTCGCGAGAG CAAGCGGACC TCATAAAGTG 950 951 CGTCGTAGTC CGGATTGGAG TCTGCAACTC GACTCCATGA AGTCGGAATC 1000 1001 GCTAGTAATC GTGGATCAGA ATGCCACGGT GAATACGTTC CCGGGCCTTG 1050 1051 TACACACCGC CCGTCACACC ATGGGAGTGG G 1081 PL 3.2 Trình t o n gen 16S rDNA c a ch ng H18 (KC894666) ACGGGAGGCA GCAGTGGGGA ATATTGCACA ATGGGCGCAA GCCTGATGCA 50 51 GCCATGCCGC GTGTATGAAG AAGGCCTTCG GGTTGTAAAG TACTTTCAGC 100 101 GGGGAGGAAG GTGTTGTGGT TAATAACCAC AGCAATTGAC GTTACCCGCA 150 151 GAAGAAGCAC CGGCTAACTC CGTGCCAGCA GCCGCGGTAA TACGGAGGGT 200 201 GCAAGCGTTA ATCGGAATTA CTGGGCGTAA AGCGCACGCA GGCGGTCTGT 250 251 TAAGTCAGAT GTGAAATCCC CGGGCTCAAC CTGGGAACTG CATTTGAAAC 300 301 TGGCAGGCTT GAGTCTCGTA GAGGGGGGTA GAATTCCAGG TGTAGCGGTG 350 351 AAATGCGTAG AGATCTGGAG GAATACCGGT GGCGAAGGCG GCCCCCTGGA 400 401 CGAAGACTGA CGCTCAGGTG CGAAAGCGTG GGGAGCAAAC AGGATTAGAT 450 451 ACCCTGGTAG TCCACGCCGT AAACGATGTC GACTTGGAGG TTGTGCCCTT 500 501 GAGGCGTGGC TTCCGGAGCT AACGCGTTAA GTCGACCGCC TGGGGAGTAC 550 551 GGCCGCAAGG TTAAAACTCA AATGAATTGA CGGGGGCCCG CACAAGCGGT 600 601 GGAGCATGTG GTTTAATTCG ATGCAACGCG AAGAACCTTA CCTGGTCTTG 650 651 ACATCCAGAG AATCCTGCAG AGATGCGGGA GTGCCTTCGG GAACTCTGAG 700 701 ACAGGTGCTG CATGGCTGTC GTCAGCTCGT GTTGTGAAAT GTTGGGTTAA 750 751 GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG GTCCGGCCGG 800 801 GAACTCAAAG GAGACTGCCG GTGATAAACC GGAGGAAGGT GGGGATGACG 850 851 TCAAGTCATC ATGGCCCTTA CGACCAGGGC TACACACGTG CTACAATGGC 900 901 GCATACAAAG AGAAGCGACC TCGCGAGAGC AAGCGGACCT CATAAAGTGC 950 951 GTCGTAGTCC GGATTGGAGT CTGCAACTCG ACTCCATGAA GTCGGAATCG 1000 1001 CTAGTAATCG TGGATCAGAA TGCCACGGTG AATACGTTCC CGGGCCTTGT 1050 1051 ACACACCGCC CGT - - 1063 PL 3.3 Trình t gen 16S rDNA c a ch ng H51 (KC894667) CGGGAGGCAG CAGTGGGGAA TATTGCACAA TGGGCGCAAG CCTGATGCAG 50 51 CCATGCCGCG TGTATGAAGA AGGCCTTCGG GTTGTAAAGT ACTTTCAGCG 100 101 GGGAGGAAGG TGTTGTGGTT AATAACCACA GCAATTGACG TTACCCGCAG 150 151 AAGAAGCACC GGCTAACTCC GTGCCAGCAG CCGCGGTAAT ACGGAGGGTG 200 201 CAAGCGTTAA TCGGAATTAC TGGGCGTAAA GCGCACGCAG GCGGTCTGTT 250 251 AAGTCAGATG TGAAATCCCC GGGCTCAACC TGGGAACTGC ATTTGAAACT 300 301 GGCAGGCTTG AGTCTCGTAG AGGGGGGTAG AATTCCAGGT GTAGCGGTGA 350 351 AATGCGTAGA GATCTGGAGG AATACCGGTG GCGAAGGCGG CCCCCTGGAC 400 401 GAAGACTGAC GCTCAGGTGC GAAAGCGTGG GGAGCAAACA GGATTAGATA 450 451 CCCTGGTAGT CCACGCCGTA AACGATGTCG ACTTGGAGGT TGTGCCCTTG 500 501 AGGCGTGGCT TCCGGAGCTA ACGCGTTAAG TCGACCGCCT GGGGAGTACG 550 551 GCCGCAAGGT TAAAACTCAA ATGAATTGAC GGGGGCCCGC ACAAGCGGTG 600 601 GAGCATGTGG TTTAATTCGA TGCAACGCGA AGAACCTTAC CTGGTCTTGA 650 651 CATCCAGAGA ATCCTGCAGA GATGCGGGAG TGCCTTCGGG AACTCTGAGA 700 701 CAGGTGCTGC ATGGCTGTCG TCAGCTCGTG TTGTGAAATG TTGGGTTAAG 750 751 TCCCGCAACG AGCGCAACCC TTATCCTTTG TTGCCAGCGG TCCGGCCGGG 800 801 AACTCAAAGG AGACTGCCGG TGATAAACCG GAGGAAGGTG GGGATGACGT 850 851 CAAGTCATCA TGGCCCTTAC GACCAGGGCT ACACACGTGC TACAATGGCG 900 901 CATACAAAGA GAAGCGACCT CGCGAGAGCA AGCGGACCTC ATAAAGTGCG 950 951 TCGTAGTCCG GATTGGAGTC TGCAACTCGA CTCCATGAAG TCGGAATCGC 1000 1001 TAGTAATCGT GGATCAGAAT GCCACGGTGA ATACGTTCCC GGGCCTTGTA 1050 1051 CACACCGCCC GT - 1062 PL 3.4 Trình t o n gen 16S rDNA c a ch ng H61 (KC894667) ACGGGAGGCA GCAGTGGGGA ATATTGCACA ATGGGCGCAA GCCTGATGCA 50 51 GCCATGCCGC GTGTATGAAG AAGGCCTTCG GGTTGTAAAG TACTTTCAGC 100 101 GGGGAGGAAG GTGTTGTGGT TAATAACCAC AGCAATTGAC GTTACCCGCA 150 151 GAAGAAGCAC CGGCTAACTC CGTGCCAGCA GCCGCGGTAA TACGGAGGGT 200 201 GCAAGCGTTA ATCGGAATTA CTGGGCGTAA AGCGCACGCA GGCGGTCTGT 250 251 TAAGTCAGAT GTGAAATCCC CGGGCTCAAC CTGGGAACTG CATTTGAAAC 300 301 TGGCAGGCTT GAGTCTCGTA GAGGGGGGTA GAATTCCAGG TGTAGCGGTG 350 351 AAATGCGTAG AGATCTGGAG GAATACCGGT GGCGAAGGCG GCCCCCTGGA 400 401 CGAAGACTGA CGCTCAGGTG CGAAAGCGTG GGGAGCAAAC AGGATTAGAT 450 451 ACCCTGGTAG TCCACGCCGT AAACGATGTC GACTTGGAGG TTGTGCCCTT 500 501 GAGGCGTGGC TTCCGGAGCT AACGCGTTAA GTCGACCGCC TGGGGAGTAC 550 551 GGCCGCAAGG TTAAAACTCA AATGAATTGA CGGGGGCCCG CACAAGCGGT 600 601 GGAGCATGTG GTTTAATTCG ATGCAACGCG AAGAACCTTA CCTGGTCTTG 650 651 ACATCCAGAG AATCCTGCAG AGATGCGGGA GTGCCTTCGG GAACTCTGAG 700 701 ACAGGTGCTG CATGGCTGTC GTCAGCTCGT GTTGTGAAAT GTTGGGTTAA 750 751 GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG GTCCGGCCGG 800 801 GAACTCAAAG GAGACTGCCG GTGATAAACC GGAGGAAGGT GGGGATGACG 850 851 TCAAGTCATC ATGGCCCTTA CGACCAGGGC TACACACGTG CTACAATGGC 900 901 GCATACAAAG AGAAGCGACC TCGCGAGAGC AAGCGGACCT CATAAAGTGC 950 951 GTCGTAGTCC GGATTGGAGT CTGCAACTCG ACTCCATGAA GTCGGAATCG 1000 1001 CTAGTAATCG TGGATCAGAA TGCCACGGTG AATACGTTCC CGGGCCTTGT 1050 1051 ACACACCGCC CGTCACACCA TGGGAGTGGG - 1080 PL 3.5 Trình t o n gen 16S rDNA c a ch ng H77 (KC894669) TACGGGAGGC AGCAGTGGGG AATATTGCAC AATGGGCGCA AGCCTGATGC 50 51 AGCCATGCCG CGTGTATGAA GAAGGCCTTC GGGTTGTAAA GTACTTTCAG 100 101 CGGGGAGGAA GGTGTTGTGG TTAATAACCG CAGCAATTGA CGTTACCCGC 150 151 AGAAGAAGCA CCGGCTAACT CCGTGCCAGC AGCCGCGGTA ATACGGAGGG 200 201 TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG 250 251 TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA 300 301 CTGGCAGGCT GGAGTCTTGT AGAGGGGGGT AGAATTCCAG GTGTAGCGGT 350 351 GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG 400 401 ACAAAGACTG ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA 450 451 TACCCTGGTA GTCCACGCCG TAAACGATGT CGATTTGGAG GTTGTGCCCT 500 501 TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA 550 551 CGGCCGCAAG GTTAAAACTC AAATGAATTG ACGGGGGCCC GCACAAGCGG 600 601 TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGTCTT 650 651 GACATCCACA GAACTTTCCA GAGATGGATT GGTGCCTTCG GGAACTGTGA 700 701 GACAGGTGCT GCATGGCTGT CGTCAGCTCG TGTTGTGAAA TGTTGGGTTA 750 751 AGTCCCGCAA CGAGCGCAAC CCTTATCCTT TGTTGCCAGC GGTTAGGCCG 800 801 GGAACTCAAA GGAGACTGCC AGTGATAAAC TGGAGGAAGG TGGGGATGAC 850 851 GTCAAGTCAT CATGGCCCTT ACGACCAGGG CTACACACGT GCTACAATGG 900 901 CGCATACAAA GAGAAGCGAC CTCGCGAGAG CAAGCGGACC TCATAAAGTG 950 951 CGTCGTAGTC CGGATTGGAG TCTGCAACTC GACTCCATGA AGTCGGAATC 1000 1001 GCTAGTAATC GTAGATCAGA ATGCTACGGT GAATACGTTC CCGGGCCTTG 1050 1051 TACACACCGC CCGT - 1064 PL 3.6 Trình t o n gen 16S rDNA c a ch ng H108 (KC894670) ACGGGAGGCA GCAGTGGGGA ATATTGCACA ATGGGCGCAA GCCTGATGCA 50 51 GCCATGCCGC GTGTATGAAG AAGGCCTTCG GGTTGTAAAG TACTTTCAGC 100 101 GGGGAGGAAG GTGTTGTGGT TAATAACCAC AGCAATTGAC GTTACCCGCA 150 151 GAAGAAGCAC CGGCTAACTC CGTGCCAGCA GCCGCGGTAA TACGGAGGGT 200 201 GCAAGCGTTA ATCGGAATTA CTGGGCGTAA AGCGCACGCA GGCGGTCTGT 250 251 TAAGTCAGAT GTGAAATCCC CGGGCTCAAC CTGGGAACTG CATTTGAAAC 300 301 TGGCAGGCTT GAGTCTCGTA GAGGGGGGTA GAATTCCAGG TGTAGCGGTG 350 351 AAATGCGTAG AGATCTGGAG GAATACCGGT GGCGAAGGCG GCCCCCTGGA 400 401 CGAAGACTGA CGCTCAGGTG CGAAAGCGTG GGGAGCAAAC AGGATTAGAT 450 451 ACCCTGGTAG TCCACGCCGT AAACGATGTC GACTTGGAGG TTGTGCCCTT 500 501 GAGGCGTGGC TTCCGGAGCT AACGCGTTAA GTCGACCGCC TGGGGAGTAC 550 551 GGCCGCAAGG TTAAAACTCA AATGAATTGA CGGGGGCCCG CACAAGCGGT 600 601 GGAGCATGTG GTTTAATTCG ATGCAACGCG AAGAACCTTA CCTGGTCTTG 650 651 ACATCCAGAG AATCCTGCAG AGATGCGGGA GTGCCTTCGG GAACTCTGAG 700 701 ACAGGTGCTG CATGGCTGTC GTCAGCTCGT GTTGTGAAAT GTTGGGTTAA 750 751 GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG GTCCGGCCGG 800 801 GAACTCAAAG GAGACTGCCG GTGATAAACC GGAGGAAGGT GGGGATGACG 850 851 TCAAGTCATC ATGGCCCTTA CGACCAGGGC TACACACGTG CTACAATGGC 900 901 GCATACAAAG AGAAGCGACC TCGCGAGAGC AAGCGGACCT CATAAAGTGC 950 951 GTCGTAGTCC GGATTGGAGT CTGCAACTCG ACTCCATGAA GTCGGAATCG 1000 1001 CTAGTAATCG TGGATCAGAA TGCCACGGTG AATACGTTCC CGGGCCTTGT 1050 1051 ACACACCGCC C - - 1061 PL 3.7 Trình t o n gen rpoB c a ch ng H77 (KF028398) GGATATGATC AACGCCAAGC CGATTTCTGC AGCCGTGAAA GAGTTCTTCG 50 51 GTTCCAGCCA GCTGTCTCAG TTTATGGACC AGAACAACCC GCTGTCTGAG 100 101 ATTACGCACA AACGTCGTAT CTCTGCACTC GGCCCAGGCG GTCTGACCCG 150 151 TGAACGCGCA GGCTTCGAAG TTCGAGACGT ACACCCGACT CACTACGGTC 200 201 GCGTATGTCC AATCGAAACG CCTGAAGGTC CAAACATCGG TCTGATCAAC 250 251 TCCCTGTCCG TGTACGCACA GACAAACGAA TACGGTTTCC TTGAGACCCC 300 301 GTACCGTAAA GTGACTGACG GTGTTGTTAC CGACGAAATT CATTACCTGT 350 351 CTGCTATTGA AGAAGGCAAC TACGTTATCG CTCAGGCGAA CTCCAACCTG 400 401 GATGACGAAG GCCACTTTGT AGAAGATCTG GTTACCTGCC GTAGCAAAGG 450 451 CGAATCCAGC TTGTTCAGCC GCGACCAGGT TGACTACATG GACGTATCCA 500 501 CCCAGCAGGT GGTATCCGTC GGTGCGTCCC TGATCCCGTT CCTGGAACAC 550 551 GATGACGCCA ACCGTGCATT GATGGGTGCG AACATGCAAC GTCAGGCCGT 600 601 TCCAACTCTG CGTGCTGATA AGCCGCTGGT TGGTACCGGT ATGGAACGTG 650 651 CTGTTGCCGT TGACTCCGGT GTTACTGCAG TGGCTAAGCG TGGCGGTACC 700 701 GTTCAGTACG TTGACGCATC CCGTATCGTT ATCAAAGTTA ACGAAGACGA 750 751 GATGTATCCG GGCGAAGCGG GTATCGACAT CTACAACCTG ACCAAATACA 800 801 CCCGTTCTAA CCAGAACACC TGTATCAACC AGATGCCTTG CGTATCTCTG 850 851 GGTGAGCCAG TTGAGCGCGG CGACGTGCTG GCAGACGGTC CGTCCACCGA 900 901 CCTC 904 PL 3.8 Trình t o n gen rpoB c a ch ng H108 (KF028399) TCCGCGGCGG TGAAAGAGTT CTTCGGTTCA AGCCAGCTTT CTCAGTTTAT 50 51 GGACCAGAAC AACCCGCTGT CTGAGATCAC GCACAAGCGT CGTATCTCTG 100 101 CACTCGGCCC GGGCGGTCTG ACGCGTGAGC GCGCAGGCTT CGAAGTTCGA 150 151 GACGTTCACC CGACCCACTA CGGTCGCGTA TGTCCTATCG AAACGCCGGA 200 201 AGGTCCGAAC ATCGGTCTGA TCAACTCCCT GTCCGTGTAC GCACAGACTA 250 251 ACGAGTATGG CTTCCTTGAG ACGCCGTACC GTAAAGTGAC CGACGGTGTG 300 301 GTGACTGATG AAATTCATTA CCTCTCCGCT ATCGAAGAAG GTAACTACGT 350 351 TATCGCGCAG GCGAACACCA ACCTGACCGA AGAAGGCCGC TTCGCCGATG 400 401 ATTTGGTCAC CTGCCGCAGC AAAGGCGAAT CCAGCCTCTT CAGCGCAGAC 450 451 CAGGTTGACT ATATGGACGT TTCCACCCAG CAGGTGGTTT CTGTCGGTGC 500 501 GTCCCTGATC CCGTTCCTTG AGCATGATGA CGCCAACCGT GCATTGATGG 550 551 GTGCGAACAT GCAACGTCAG GCGGTTCCGA CCCTGCGCGC TGATAAGCCG 600 601 CTGGTAGGTA CCGGTATGGA ACGTGCTGTT GCCGTTGACT CCGGTGTAAC 650 651 CGCCGTGGCC AAACGTGGCG GTATCGTTCA GTACGTGGAT GCGTCCCGTA 700 701 TCGTTATCAA AGTTAACGAA GACGAGATGT ACCCGGGCGA AGCAGGTATC 750 751 GACATCTATA ACCTGACTAA ATACACCCGT TCTAACCAGA ACACCTGCAT 800 801 CAACCAGATG CCGTGCGTGT CTCTGAATGA ACCGGTTGAG CGCGGCGACG 850 851 TGCTGGC - 857 ... m vi sinh nuôi tr ng h i s n, t vi khu n có kh sinh bacteriocin, phù h p v i c i m mơi trư ng i u ki n khí h u ? ?Phân l p n ch n vi khu n sinh bacteriocin t s ng Vi t Nam, tài ng v t thân m m hai. .. t qu c a lu n văn ? ?Phân l p n ch n vi khu n sinh bacteriocin t ng v t thân m m hai v s ng ch n nghiên c u bi n” thu c tài ? ?Phân l p, n c i m sinh h c c a vi khu n bi n sinh bacteriocin dùng làm... th gi i v vi khu n bi n sinh bacteriocin c phân l p ch v n b ng i tư ng th y s n, cá ng v t thân m m Vi t Nam Hi n chưa có nghiên c u th gi i Vi t Nam v vi khu n bi n sinh bacteriocin phân l p