Xây dựng phương án thiết kế mồi phục vụ tách dòng gen mong muốn

Họ tên: Đoàn Ngọc Nhinh MSSV: 20175035 Lớp: KTSH.01 Mã lớp học: 694235 BÁO CÁO THÍ NGHIỆM Thực hành tin sinh học I Mục đích thí nghiệm: Xây dựng phương án thiết kế mồi phục vụ tách dòng gen mong muốn II Thao tác tiến hành: Xác định gene cần tách: - Xác định tính trạng quan tâm - Xác định gene quy định tính trạng báo cáo khoa học - Tìm kiếm số gene mã hóa protein quan tâm ngân hàng liệu Thiết kế mồi: - Align trình tự mã hóa protein Clustal Omega - Xác định vùng bảo thủ (mồi thiết kế nằm vùng bảo thủ này) - Thiết kế mồi: sử dụng chương trình như: Primer Blast, Primer3Plus, Primer3 - Điều kiện mồi: • độ dài: 18-24 bp • Nhiệt độ bắt mồi nhiệt k lệch 5oC • Tỉ lệ GC: 40-60% Tối ưu 50-55% • nu cuối đầu 3’ cần có it G C • Hạn chế khả tự bắt cặp mồi bắt cặp mồi với nhau: hạn chế đoạn lặp đoạn từ nu trùng trở lên Kiểm tra mồi: Sử dụng PCR ảo SMS Xem xét đoạn kết sau chạy SMS xem có tương đương (kích thước,… ) với đoạn mà định nhân lên không, cho vào Blast ncbi để xem có gen mã hóa tính trạng cần khơng Phân tích cấu trúc 3D, dự đốn sản phẩm tách dịng biểu protein Xây dựng phương án tách dòng gene III Kết thực hành: Xác định gene cần tách: - Tính trạng quan tâm: khả chịu hạn ngơ Lý do: Tình hình sản xuất ngơ nước ta chưa tương xứng với tiềm năng, chưa đáp ứng nhu cầu nước, phải nhập ngơ từ nước ngồi Do đa số ngơ nước ta trồng khu vực miền núi, tình hình biến đổi khí hậu dẫn đến việc khơ hạn ngày diễn kéo dài, phức tạp hơn, dẫn tới không đảm bảo lượng nước theo nhu cầu ngơ - Gene quy định tính trạng: ZmDREB2A - Truy cập sở liệu NCBI: https://www.ncbi.nlm.nih.gov/nuccore/?term=ZmDREB2A - Từ sở liệu NCBI xác định gene: AB218832.1, XM_023300797.1, XM_008657225.2, XM_008657227.2, XM_020542336.1, NM_001305945.1, NM_001305944.1, NM_001112406.2, JF915836.1 Thiết kế mồi: - Kết sau sử dụng Clustal Omega: https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20200623-1733390210-74199168-p1m AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 -TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 56 56 56 56 60 60 60 60 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 -GGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA ***************************************************** 53 116 116 116 116 120 120 120 120 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA ************************************************************ 113 176 176 176 176 180 180 180 180 AB218832.1 XM_023300797.1 XM_008657225.2 TGCCGATGCAGCCCCCGGCCCTG TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAG -TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG 136 210 236 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAG -TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAG -*********************** 236 236 240 240 214 214 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 TGGTGCATGGTTCCATCAGAACAGTAGCAGGAGGCCCTACTCTTGCCTTGAATGAGTGCC TGGTGCATGGTTCCATCAGAACAGTA -GGAGGCCCTACTCTTGCCTTGAATGAGTGCC TGGTGCATGGTTCCATCAGAACAGTAGCAG -TGGTGCATGGTTCCATCAGAACAGTAGG -TGGTGCATGGTTCCATCAGAACAGTAGCAGGAGGCCCTACTCTTGCCTTGAATGAGTGCC - 136 210 296 293 266 268 300 214 214 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CAGCCCGGAA CA AGATTTTGGTACAGCAGAAACCCCAGGGAGATTCACGACTGCTGGCCTCTAATCTGTGCA AGATTTTGGTACAGCAGAAACCCCAGGGAGATTCACGACTGCTGGCCTCTAATCTGTGCA A AGATTTTGGTACAGCAGAAACCCCAGGGAGATTCACGACTGCTGGCCTCTAATCTGTGGA GA GA 146 212 356 353 266 269 360 216 216 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC -GAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC ********************************************************* 206 272 416 413 325 326 417 273 273 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA ************************************************************ 266 332 476 473 385 386 477 333 333 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GAAAAGCACCTGCAAAGGGTTCGAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA ********************** ************************************* 326 392 536 533 445 446 537 393 393 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA ************************************************************ 386 452 596 593 505 506 597 453 453 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 446 512 656 653 565 566 NM_001305944.1 NM_001112406.2 JF915836.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG ************************************************************ 657 513 513 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CAGCTAGGGCCTATGACGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT **************** ******************************************* 506 572 716 713 625 626 717 573 573 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTAGCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG ***************************************** ****************** 566 632 776 773 685 686 777 633 633 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CTGCTCCAACAGCTGTCGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT **************** ******************************************* 626 692 836 833 745 746 837 693 693 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CAAATTACTCGGACATCGCATCCACCTCACACAAGCCTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT ***** ****************************** *********************** 686 752 896 893 805 806 897 753 753 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CCCTAAAGGCAAAATGCCCAGCTGGATCATGTGGTATCCAAGAGGGTACACCCAGTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG ******** ****** ************************** ** ******* ***** 746 812 956 953 865 866 957 813 813 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG ************************************************************ 806 872 1016 1013 925 926 1017 873 873 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 866 932 1076 1073 985 986 1077 933 933 ************************************************************ AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 CTGACGCTGGCATGGGGCAGCCCTGGTGTCTTGATGAGCTGGATTCGAGTGTCTTGGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA * ************** ********** *************************** **** 926 992 1136 1133 1045 1046 1137 993 993 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GCATGCTCCAGCCACAGC CAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG ************* *** ************************************ 980 1052 1196 1193 1105 1106 1197 1053 1053 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT ************************************************************ 1040 1112 1256 1253 1165 1166 1257 1113 1113 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG ************************************************************ 1100 1172 1316 1313 1225 1226 1317 1173 1173 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTTATATTCCTCTGT GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG *********************************************** * * 1160 1230 1374 1371 1283 1284 1375 1231 1231 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 AAATAGTTCTCTAGTCATGGGAACTGGGTTTGCTTCACATTTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG *********** ************************ ******************** 1220 1290 1434 1431 1343 1344 1435 1291 1291 AB218832.1 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC ************************************************************ 1280 1350 1494 1491 1403 1404 1495 1351 1351 AB218832.1 TTT - 1283 XM_023300797.1 XM_008657225.2 XM_008657227.2 XM_020542336.1 NM_001305945.1 NM_001305944.1 NM_001112406.2 JF915836.1 TTTCGTTCCTTA TTTCGTTCCTTA TTTCGTTCCTTA TTTCGTTCCTTA TTTCGTTCCTTA TTTCGTTCCTTA TTTCGTTCCTTA TTTC 1362 1506 1503 1415 1416 1507 1363 1355 *** Từ kết quả, nhận thấy gene NM_001305944.1 (1507 bp) chứa nhiều trình tự bảo thủ, độ dài lớn, gene ngô Trung Quốc, nên có khả tương đồng với gene ngô Việt Nam cần nghiên cứu Đoạn bảo thủ: (nhiều dấu sao): Vị trí 68-203 1272-1510 - Chạy Primer Blast: truy cập trang web: https://www.ncbi.nlm.nih.gov/tools/primer-blast/ Cài đặt thông số: Forward primer: 68-203 Reverse primer: 1272-1498 PCR product size: 70-1510 Kết quả: Từ kết lựa chọn mồi Primer Pair 4có: mồi xi gần đầu trình tự gene gốc mồi ngược gần cuối gene gốc, có tỉ lệ Self complementarity tương đối thấp, nhiệt độ bắt cặp thích hợp, tỉ lệ GC% nằm mức tối ưu… Forward primer GAACACACTACACACCAGCC Reverse primer GCAAACCCAGTTCCCATGC Kiểm tra mồi: • Kết sau chạy PCR ảo trang web: https://www.bioinformatics.org/sms2/pcr_products.html PCR Products results >1321 bp product from linear template NM_001305944.1 Zea mays DRE-binding protein 1c (LOC732788), transcript variant 1, mRNA, base 88 to base 1408 (T7 - T3) GAACACACTACACACCAGCCAGCGAGATAGCGAACGCTAGGAACCCAGTGGCCATCTTTG GAGCGGCCATGACGCTGGATCAGAACCATGCCATGCCGATGCAGCCCCCGGCCCTGCAGC CCGGAAGAGCATATGGAGCAGAGGGCAGTGCTGTGGTGCATGGTTCCATCAGAACAGTAG CAGGAGGCCCTACTCTTGCCTTGAATGAGTGCCAGATTTTGGTACAGCAGAAACCCCAGG GAGATTCACGACTGCTGGCCTCTAATCTGTGGAAGAAGCGACCTCGCAGATCACGAGATG GGCCTACGTCAGTGGCAGCTGTCATCCAGCGGTGGGCTGAGCGCAACAAGCATTTGGAGT ATGAGGAATCTGAGGAGGCAAAGCGACCAAGAAAAGCACCTGCAAAGGGTTCAAAGAAGG GCTGTATGAAGGGAAAAGGGGGGCCTGACAATACTCAATGTGGATACCGTGGAGTGAGGC AGCGTACTTGGGGGAAGTGGGTTGCTGAAATAAGAGAGCCAAATCGTGTCGACAGACTCT GGCTGGGTACCTTCCCAACCGCGGAGGATGCAGCTAGGGCCTATGATGAGGCAGCCAGAG CGATGTATGGAGACTTGGCACGGACTAACTTCCCCGGACAGGATGCAACAACCTCTGCCC AAGCTGCTCTATCATCGACCTCTGCCCAGGCTGCTCCAACAGCTGTTGAAGCTCTTCAGA CTGGCACGTCATGCGAGTCGACAACGACATCAAATCACTCGGACATCGCATCCACCTCAC ACAAGCTTGAAGCCTCTGACATCTCGAGCTACCTAAAGGAAAAATGTCCAGCTGGATCAT GTGGTATCCAAGACGGCACACCCATTGTAGCTGACAAGGAGGTCTTTGGGCCGTTGGAGC CTATCACAAATCTTCCAGATGGTGGTGATGGTTTTGATATCGGTGAGATGCTGAGGATGA TGGAAAGCGATCCACATAATGCAGGTGGAGCCGACGCTGGCATGGGACAGCCCTGGTATC TTGATGAGCTGGATTCGAGTGTCTTAGAGAGCATGCTCCAGCCGGAGCCGGAGCCAGAGC CAGAGCCATTCCTGATGTCTGAAGAACCGGACATGTTTCTTGCTGGCTTCGAAAGCGCTG GTTTCGTCGAGGGTCTGGAGCGGCTAAACTGAATTTCTGATGTTTGACCGTTGATCCTCA TCCCACTTCATGTCTGAGCTTGTGAATTCGGAGGCAAACATTGGCAGAACTTATAAGCTC TAGCAATTCTAGGCTTTACATTCCTCTGTGATAGTTCTCTATGCATGGGAACTGGGTTTG C Nhận thấy đoạn kết có kích thước tương đương với đoạn gene định nhân lên • So sánh tương đồng mạch ảo so với chuỗi tương ứng lưu trữ ngân hàng gen Clustal Omega: - Tương tự so sánh trình tự PCR test với gene khác cho thấy tương đồng lớn => Đoạn bảo thủ lựa chọn tương đối phù hợp • Đối chiếu lên Blast: https://blast.ncbi.nlm.nih.gov/Blast.cgi Chọn Nucleotide => Protein Thu kết quả: Protein tương đồng cao với mục tiêu: >NP_001292873.1 dehydration-responsive element binding protein 2A isoform [Zea mays] MTLDQNHAMPMQPPALQPGRAYGAEGSAVVHGSIRTVAGGPTLALNECQILVQQKPQGDSRLLASNLWKK RPRRSRDGPTSVAAVIQRWAERNKHLEYEESEEAKRPRKAPAKGSKKGCMKGKGGPDNTQCGYRGVRQRT WGKWVAEIREPNRVDRLWLGTFPTAEDAARAYDEAARAMYGDLARTNFPGQDATTSAQAALSSTSAQAAP TAVEALQTGTSCESTTTSNHSDIASTSHKLEASDISSYLKEKCPAGSCGIQDGTPIVADKEVFGPLEPIT NLPDGGDGFDIGEMLRMMESDPHNAGGADAGMGQPWYLDELDSSVLESMLQPEPEPEPEPFLMSEEPDMF LAGFESAGFVEGLERLN Phân tích cấu trúc 3D, dự đốn sản phẩm tách dịng biểu protein • Dự đoán pI, khối lượng phân tử: Truy cập https://web.expasy.org/protparam/ Nhập trình tự protein vào kết quả: pI= 5.02 MW= 39577.11 Da • Dự đốn chức năng: - Truy cập: https://www.uniprot.org/uniref/ - Vào mục Blast, nhập vào trình tự PCR ảo - Chọn protein có độ tương đồng cao vào mục Funtion: => Chức năng: - Yếu tố phiên mã hoạt động gắn DNA - Liên kết DNA đặc hiệu - Phiên mã liên kết DNA khu vực quy định • Tìm vị trí: - Truy cập: http://phobius.sbc.su.se/index.html - Nhập vào trình tự protein => Enzyme ngoại bào • Cấu trúc không gian chiều: -Trên trang Blast protein, ý tới region name - tra NCBI với miền Structure : AP2: - Dowload máy dùng phần mềm Cn3D để mở - Vào Style=>Coloring Shortcuts => Secondary Structure Xây dựng phương án tách dòng gene: 5.1 Vật liệu nghiên cứu: - Vector tách dịng pBluescript - Vector chuyển gen ubi::ZmDREB2A::35S - Giống ngơ ưu tú chọn lọc để tách dòng gene: B163, P4092, PAC746, TG8327, X7A305 5.2 Phương pháp nghiên cứu: *Tách chiết RNA tổng số khuếch đại gene từ cDNA - RNA tổng số tách từ nụ hoa ngô ưu tú - cDNA sau tổng hợp sử dụng làm khuôn cho phản ứng PCR khuếch đại gen ZmDREB2A với cặp mồi đặc hiệu - Sản phẩm PCR kiểm tra gel agarose tinh *Gắn vào vector tách dòng - Sử dụng vector nhân dòng pBluescript - Sản phẩm PCR nhân gen ZmDREB2A vector tách dòng pBluescript cắt đồng thời hai enzyme đặc hiệu - Sản phẩm cắt sau tinh ghép nối enzyme T4 ligase - Sản phẩm ghép nối sau biến nạp vào tế bào vi khuẩn Agrobacterium tumefaciens khả biến theo phương pháp sốc nhiệt 42oC *Chọn lọc dòng tế bào tái tổ hợp - Tế bào tái tổ hợp sàng lọc PCR với cặp mồi đặc hiệu từ khuẩn lạc - Tế bào tái tổ hợp nuôi tăng sinh, tách plasmid để kiểm tra đoạn chèn enzyme cắt hạn chế giải trình tự gen - Trình tự gen sau kiểm tra cách so sánh với trình tự gen gốc ngân hàng gen NCBI công cụ BLAST *Gắn vào vector chuyển gen - Vector biểu ubi::ZmDREB2A::35S sử dụng để chuyển gen ZmDREB2A vào trồng.Vector ubi::ZmDREB2A::35S cắt đồng thời enzyme đặc hiệu sauđó ghép nối enzyme T4 DNA ligase - Sản phẩm ghép nối chuyển vào tế bào vi khuẩn A tumefaciens nuôi cấy môi trường LB - Để chuyển vào trồng, plasmid tái tổ hợp chuyển vào tế bào vi khuẩn  ... Structure Xây dựng phương án tách dòng gene: 5.1 Vật liệu nghiên cứu: - Vector tách dịng pBluescript - Vector chuyển gen ubi::ZmDREB2A::35S - Giống ngơ ưu tú chọn lọc để tách dòng gene: B163,... Reverse primer: 1272-1498 PCR product size: 70-1510 Kết quả: Từ kết lựa chọn mồi Primer Pair 4có: mồi xi gần đầu trình tự gene gốc mồi ngược gần cuối gene gốc, có tỉ lệ Self complementarity tương đối... ZmDREB2A với cặp mồi đặc hiệu - Sản phẩm PCR kiểm tra gel agarose tinh *Gắn vào vector tách dòng - Sử dụng vector nhân dòng pBluescript - Sản phẩm PCR nhân gen ZmDREB2A vector tách dòng pBluescript