Luận án Nghiên cứu biểu hiện protein tái tổ hợp miraculin trong dòng tế bào BY2, rễ tơ thuốc lá và cây cà chua chuyển gen

ell and hairy root was observed in transformants with HSP-pro/Mir/HSP-ter cassette. Modified miraculin gene was has been also transferred into tomato plants with transformation high rate reached from 42.11% to 52.38 %. Trasgenic tomato lines were comfirmed by PCR and Southern blot analysis for 27 lines transgenic tomato plants in which 16 lines are transferred E8-pro/Mir/HSP-ter and 11 35S- pro/Mir/NOS-ter casstte, there are 5 transgenic tomato lines carrying single copy (TE8.2, TE8.7, TE8.8, T35S.2, T35S8). For 11 transgenic tomato lines carrying 35S-pro/Mir/NOS-ter cassette, expression genes miraculin in mRNA levels were also confirmed by RT-PCR analysis. Recombinant miraculin content in the red fruit exocarp of E8-pro/Mir/HSP-ter (T.E8.8) reached 118,0 ng/µg in total solute protein and was approximately 4,0 times higher than that in the 35S-pro/Mir/NOS-ter transgenic line. Furthermore, these transgenic tomato are expressed system to produce recombinant miraculin in the agriculture. 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Cell 115(3):255- 266. 152. Zuo J, Chua N (2000) Chemical-inducible systems for regulated expression of plant genes. Curr Opin Biotechnol 11:146-151. PHỤ LỤC 1. So sánh trình tự nucleotide của promoter E8 thu đƣợc với trình tự AF515784.1 và DQ317599.1 10 20 30 40 50 60 70 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ---------- AAGCTTTCCC TAATGATATT GTTCATGTAA TTAAGTTTTG TGGAAGTGAG AGAGTCCAAT AF515784.1 ---------- ------.... .......... .......... .......... .......... .......... DQ317599.1 GAATTCATTT TT.ACA.... .......... .....C.... .......... .......... .......... 80 90 100 110 120 130 140 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTGATAAGA AAAGAGTCAG AAAACGTAAT ATTTTAAAAG TCTAAATCTT TCTACAAATA AGAGCAAATT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 150 160 170 180 190 200 210 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TATTTATTTT TTAATCCAAT AAATATTAAT GGAGGACAAA TTCAATTCAC TT-GGTTGTA AAATAAACTT AF515784.1 .......... .......... .......... .......... .......... ..T....... .......... DQ317599.1 .......... .......... .......... .......... .......... ..-....... .......... 220 230 240 250 260 270 280 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AAACCAATAA CCAAAGAACT AATAAATCCT GAAGTGGAAT TATTAAGGAT AAATGTACAT AGACAATGAA AF515784.1 .......... .......... .......... .......... .......... .......... ....G..... DQ317599.1 .......... .......N.. ........-. .......... .......... ..-....... .......... 290 300 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter GAAATAATAG GTTCGATGAA TTAATAATAA TTAAGGATGT TACAATCATC ATGTGCCAAG TATATACACA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 360 370 380 390 400 410 420 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATATTCTATG GGATTTATAA TTTCGTTACT TCACTTAACT TTTGCGTAAA TAAAACGAAT TATCTGATAT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 430 440 450 460 470 480 490 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTATAATAA AACAGTTAAT TAAGAACCAT CATTTTTAAC AACATAGATA TATTATTTCT AATAGTTTAA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 500 510 520 530 540 550 560 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGATACTTTT AAATCTTTTA AATTTTATGT TTCTTTTAGA AAATAAAAAT TCAAAAAA-T TAAATATATT AF515784.1 .......... .......... .......... .......... .......... ........-. .......... DQ317599.1 .......... .......... .......... .......... .......... ........A. .......... 570 580 590 600 610 620 630 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TACAAAAACT ACAATCAAAC ACAACTTCAT ATATTAAAAG CAAAATATAT TTTGAAAATT TCAAGTGTCC AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 640 650 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAACAAATAA GACAAGAGGA AAATGTACGA TGAGAGACAT AAAGAGAACT AATAATTGAG GAGTCCTATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 710 720 730 740 750 760 770 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATATATAATA AAGTTTATTA GTAAACTTAA TTATTAAGGA CTCCTAAAAT ATATGATAGG AGAAAATGAA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 780 790 800 810 820 830 840 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGGTGAGAGA TATTGGAAAA CTTAATAATT AAGGATTTTA AAATATATGG TAAAAGATAG GCAAAGTATC AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... ......N... .......... .......... .......... 850 860 870 880 890 900 910 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CATTATCCCC TTTTAACTTG AAGTCTAC-T AGGCGCATGT GAAAG-TTGA TTTTTTGTCA CGTCATATAG AF515784.1 .......... .......... ........-. .......... .....-.... .......... .......... DQ317599.1 .......... .......... ........C. .......... .....G.... .......... .......... 920 930 940 950 960 970 980 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CTATAACGTA AAAAAAGAAA GTAAAATTTT TAATTTTTTT TAATATATGA CATATTTTAA ACGAAATATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 990 1000 1010 1020 1030 1040 1050 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter GGACAAAATG TAAATGAATA GTAAAGGAAA CAAAGATTAA TACTTACTTT GTAAGAATTT AAGATAAATT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1060 1070 1080 1090 1100 1110 1120 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAAAATTTAA TAGATCAACT TTACGTCTAG AAAGACCCTA TCTTAGAAGG AATTTCA-GA AATCGGCCCT AF515784.1 .......... .......... .......... ........AT ATC....... .......C.. .......... DQ317599.1 .......... .......... .......... ........AT ATC....... .......C.. .......... 1130 1140 1150 1160 1170 1180 1190 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTTCAAAAA TAACTTTTAA ATAATGAATT TTAAATTTTA AGAAATAATT TCCAATGAAT AAATGACATG AF515784.1 .A...G.... .......... .......... .......... .........A .......... .......... DQ317599.1 .A........ .......... .......... .......... .........A .......... .......... 1200 1210 1220 1230 1240 1250 1260 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAGCATTTTA CCTAAATATT TCAACTATTT TAATCCAATA TTAATTTGTT T-ATTCCCAA CAATAGAAAG AF515784.1 .......... .......... .......... .......... .......... .T........ .......... DQ317599.1 .......... .......... .......... .......... .......... .T........ .......... 1270 1280 1290 1300 1310 1320 1330 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TCTTGTGCAG ACATTTAATC TGACTTTTCC AGTACTAAAT ATTAATTTTC TGAAGATTTT CGGGTTTAGT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1340 1350 1360 1370 1380 1390 1400 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CCACAAGTTT TAGTGAGAAG TTTTGCTCAA AATTTTAGGT GAGAAGGTTT GATATTTATC TTTTGTTAAA AF515784.1 .......... .......... .......... .-........ .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1410 1420 1430 1440 1450 1460 1470 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTAATTTATC TAGGTGACTA TTATTTATTT AAGTAGAAAT TCATATCATT ACTTTTGCCA ACTTGTAGTC AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1480 1490 1500 1510 1520 1530 1540 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATAATAGGAG TAGGTGTATA TGATGAAGGA ATAAACAAGT TCAGTGAAGT GATTAAAATA AAATATAATT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1550 1560 1570 1580 1590 1600 1610 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAGGTGTACA TCAAATAAAA ACCTTAAAGT TTAGAAAGGC ACCGAATAAT TTTGCATAGA AGATATTAGT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1620 1630 1640 1650 1660 1670 1680 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AAATTTATAA AAATAAAAGA AATGTAGTTG TCAAGTTGTC TTCTTTTTTT TGGATAAAAA TAGCAGTTGG AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1690 1700 1710 1720 1730 1740 1750 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CTTATGTCAT TCTTTTACAA CCTCCATGCC ACTTGTCCAA TTGTTGACAC TTAACTAATT AGTTTGATTC Hộp CAAT AF515784.1 .......... .......... .......... .......... ..A....... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1760 1770 1780 1790 1800 1810 1820 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATGTATGAAT ACTAAATAAT TTTTTAGGAC TGACTCAAAT ATTTTTATAT TATCATAGTA ATATTTATCT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1830 1840 1850 1860 1870 1880 1890 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AATTTTTAGG ACCACTTATT ACTAAATAAT AAATTAACTA CTACTATATT ATTGTTGTGA AACAACAACG AF515784.1 .......... .......... ...T...... .......... .A........ .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1900 1910 1920 1930 1940 1950 1960 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTTGGTTGT TATGATGAAA CGTACACTAT ATCAGTATGA AAAATTCAAA ACGATTAGTA TAAATTATAT Hộp GATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1970 1980 1990 2000 2010 2020 2030 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGAAAATTTG ATATTTTTCT ATTCTTAATC AGACGTATTG GGTTTCATAT TTTAAAAAGG GACTAAACTT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 2040 2050 2060 2070 2080 2090 2100 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AGAAGAGAAG TTTGTTTGAA ACTACTTTTG TCTCTTTCTT GTTCCCATTT CTCTCTTAGA TTTCAAAAAG Hộp TATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 2110 2120 2130 2140 2150 2160 2170 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGAACTACTT TATCTCTTTC TTTGTTCACA TTTTATTTTA TTCTATTATA AATATGGCAT CCTCATATTG AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 2180 2190 2200 2210 ....|....| ....|....| ....|....| ....|....| ....|.... E8 promoter AGATTTTTAG AAATTATTCT AATCATTCAC AGTGCAAAAG AAGGGATCC AF515784.1 .......... .......... .......... .......... .-------- DQ317599.1 ........-- ---------- ---------- ---------- --------- Trình tự nền màu xanh lần lƣợt là: trình tự biểu hiện đặc hiệu ở quả (phụ thuộc ethylen): vị trí 1343-1489 và trình tự điều khiển quá trình chín của quả độc lập với ethylen: vị trí: 1943-2168. 2. So sánh trình tự nucleotide của promoter HSP 18.2 thu đƣợc với trình tự X17295.1 10 20 30 40 50 60 70 80 90 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro AAGCTTATGG TCATTTCTTC TGGTTCAAGC ATGACATGAA CAGGCAATAA ATAAGTTGAG ATTTTGATCA CAGTAACTGA TACTTGAATC Hộp CAAT Hộp GAGA X17295.1 ------.... .......... .......... .......... .......... .......... .......... .......... .......... 100 110 120 130 140 150 160 170 180 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro GAATCATTTA GATTTTTTTT TTTTTTAGTT TACTTGTTTA GTAAATATGT TGTCTATGTT TGTCACAAAA ACGTGGCTCA GTTCTTGTAT X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 190 200 210 220 230 240 250 260 270 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro ATATGGAGAC AAAAAAATCC ATTAAAAGAT TGTTGACATT CTCGGAAATT TAGTGCCAAC TGTTATTGCG AGAACTTACT ATAGTTTTCC X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 280 290 300 310 320 330 340 350 360 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro TTTGGCGAAA AGCTAATAAT CTTAAATCTT GATTTTGTCC TCTTTTCTCT GAGTTAGATT TTCTTAAATT CCACTTCCGA CCTATTAAGA X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 370 380 390 400 410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro AATGGGCTTT TGCAAAGAAG ATCCGCTTCA CTGAGCCCGT ATCTCGAAGA GGATAATACA ACAACAAAGC AAAACGGCAC GTAGTTTTAA Hộp GAGA X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 460 470 480 490 500 510 520 530 540 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro TTGTAACCAA GGATTGCATT TCGGTCTTGT TTCAACAAAC GAAACTTCCT GAAATGCCAA GAAAAATCTG GTCATTTCAC CACAGTGATC AAC GAAACTTCCT G (HSE) CCT GAAATGCCAA G (HSE) CAA GAAAAATCTG G (HSE) X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 550 560 570 580 590 600 610 620 630 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro ATTGTGTATG TGTTCTAAAG ACTCCAAGCG AAGGTTTTAG AAAAAGGAGC ATTTTCTATT CTATTCAAGA AACTCGAAGA ACATTCTCTC CTAAAG ACTCCAAG (HSE) CAAGA AACTCGAAG (HSE) CAAGCG AAGGTTTT (HSE) X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 640 650 660 670 680 690 700 710 720 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro TTCATCCTCT AACTTCCCTA TAAATATGTC CTTTGCTAAT CAGATCAAAT CAGCAGGAAA ATCAAGAACC AAAAGTCTCC CGAAAAGCAA Hộp TATA X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 730 ....|....| .. HSP 18.2 pro CGAACAGGAT CC X17295.1 ......------ HSE: heat shock elements 3. So sánh trình tự nucleotide của terminatorHSP 18.2 thu đƣợc với trình tự đã công bố 10 20 30 40 50 60 70 80 90 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 ter GAGCTCATAT GAAGATGAAG ATGAAATATT TGGTGTGTCA AATAAAAAGC TTGTGTGCTT AAGTTTGTGT TTTTTTCTTG GCTTGTTGTG HSP ter Nagaya ------.... .......... .......... .......... .......... .......... .......... .......... .......... 100 110 120 130 140 150 160 170 180 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 ter TTATGAATTT GTGGCTTTTT CTAATATTAA ATGAATGTAA GATCTCATTA TAATGAATAAACAAATGT↑TT CTATAATCCA TTGTGAATGT HSP ter Nagaya .......... .......... .......... .......... .......... .......... .......... .......... .......... 190 200 210 220 230 240 250 260 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. HSP 18.2 ter TTTGTTGGAT CTCTTCTGCA GCATATAACT ACTGTATGTG CTATGGTATG GACTATGGAA TATGATTAAA GATAAGGAAT TC HSP ter Nagaya .......... .......... .......... .......... .......... .......... .......... ......------ ↑poly A site 4. Thành phần các môi trƣờng nuôi cấy in vitro cơ bản Loại môi trƣờng Thành phần MS lỏng MS (I-V) + Sucrose 30 mg/l; pH 5,8 MS đặc MS lỏng + 7,5 g Agar CCM MS (I - V) bổ sung BAP 2 mg/l, NAA 2 mg/l, sucrose 30 g/l và 7,5g agar, pH 5,8 STM MS (I - V) bổ sung zeatin 2 mg/l, sucrose 30 mg/l và 7,5g agar, pH 5,8 RM MS (I - V) bổ sung IBA 0,3 mg/l, sucrose 15 g/l và 7,5g agar, pH = 5,8 5. Thành phần các môi trƣờng nuôi khuẩn Môi trƣờng Thành phần LB đặc 5 g/l yeast extract + 5 g/l NaCl + 10 g/l tryptone + 15 g/l bacto agar, pH = 7,0 LB lỏng 5 g/l yeast extract + 5 g/l NaCl + 10 g/l tryptone, pH = 7,0 6. Trình tự cặp mồi M13 F/R (5’→3’) M13_F: GTAAAACGACGGCCAG; M13_R: CAGGAAACAGCTATGAC 7. Sơ đồ cấu trúc vector pBI121 8. Sơ đồ cấu trúc vector pBluescript II SK 9. Phƣơng trình đƣờng chuẩn 9.1. Phương trình đường chuẩn protein (theo Braford, 1976, sử dụng BSA làm chất chuẩn) 9.2. Phương trình đường chuẩn protein scFv gắn đuôi c-myc Nồng độ chất chuẩn: 0; 1,25; 2,5; 5; 10; 20 ng/μl Nồng độ chất chuẩn: 0; 10; 25; 50; 100; 150; 200 ng/μl y = 3503.6x - 18.094 R² = 0.9924 0 250 500 750 1000 1250 1500 1750 2000 2250 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 C (µ g /µ l) OD595nm y = 26.318x - 1.6454 R² = 0.9862 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 0 0.2 0.4 0.6 0.8 1 C ( n g /μ l) OD630nm y = 281.98x - 36 R² = 0.903 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 C ( n g /μ l) OD630nm 10. Thành phần đệm chiết DNA (theo Xin và Chen, 2012) STT Nồng độ gốc Nồng độ sử dụng 1 1 M Tris-HCl (pH8,0) 50 mM 2 5 M NaCl 300 mM 3 0,5 M EDTA (pH 8,0) 20 mM 4 CTAB 4% 5 Nƣớc khử ion, khử trùng 11. Cà chua chuyển gen: giai đoạn quả xanh và quả chín đỏ