Xác định gene cần tách: - Xác định tính trạng quan tâm.. - Xác định gene quy định tính trạng đó trong các báo cáo khoa học.. - Tìm kiếm 1 số gene mã hóa protein quan tâm trên ngân hàng
Trang 1Họ và 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:
1 Xác định gene cần tách:
- Xác định tính trạng quan tâm
- Xác định gene quy định tính trạng đó trong các báo cáo khoa học
- Tìm kiếm 1 số gene mã hóa protein quan tâm trên ngân hàng dữ liệu
2 Thiết kế mồi:
- Align các trình tự mã hóa 1 protein bằng Clustal Omega
- Xác định vùng bảo thủ (mồi được thiết kế sẽ nằm trong vùng bảo thủ này)
- Thiết kế mồi: sử dụng các chương trình như: Primer Blast, Primer3Plus, Primer3
- Điều kiện mồi:
• độ dài: 18-24 bp
• Nhiệt độ bắt mồi của 2 nhiệt k lệch quá nhau 5oC
• Tỉ lệ GC: 40-60% Tối ưu là 50-55%
• 5 nu cuối cùng đầu 3’ cần có it nhất 2 G hoặc C
• Hạn chế khả năng tự bắt cặp của mồi và bắt cặp mồi với nhau: hạn chế các đoạn lặp hoặc những đoạn từ 4 nu trùng nhau trở lên
3 Kiểm tra mồi: Sử dụng PCR ảo SMS
Xem xét đoạn kết quả sau khi chạy SMS xem có tương đương (kích thước,… ) với đoạn mà định nhân lên không, rồi cho vào Blast của ncbi
để xem nó có là gen mã hóa tính trạng mình cần không
4 Phân tích cấu trúc 3D, dự đoán sản phẩm tách dòng và biểu hiện protein
5 Xây dựng phương án tách dòng gene
Trang 2III Kết quả thực hành:
1 Xác định gene cần tách:
- Tính trạng quan tâm: khả năng chịu hạn của cây ngô
Lý do: Tình hình sản xuất ngô ở nước ta chưa tương xứng với tiềm năng, chưa đáp ứng được nhu cầu trong nước, còn phải nhập khẩu ngô từ nước ngoài Do đa số ngô ở nước ta được trồng ở khu vực miền núi, tình hình biến đổi khí hậu dẫn đến việc khô hạn ngày càng diễn ra kéo dài, phức tạp hơn, dẫn tới không đảm bảo được lượng nước theo nhu cầu của cây ngô hiện tại
- Gene quy định tính trạng: ZmDREB2A
- Truy cập cơ sở dữ liệu NCBI:
https://www.ncbi.nlm.nih.gov/nuccore/?term=ZmDREB2A
- Từ cơ sở dữ liệu NCBI xác định được 9 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
2 Thiết kế mồi:
- Kết quả sau khi sử dụng Clustal Omega:
https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20200623-173339-0210-74199168-p1m
AB218832.1 - 0
XM_023300797.1 TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 56 XM_008657225.2 TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 56 XM_008657227.2 TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 56 XM_020542336.1 TTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 56 NM_001305945.1 GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 60 NM_001305944.1 GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 60 NM_001112406.2 GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 60 JF915836.1 GCTTTTGGATTCTGGTTTCTCTCGTCTTTCTCCTCCCTCTCCTGACTTTTCTTCTAGACA 60
AB218832.1 -GGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 53 XM_023300797.1 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 116 XM_008657225.2 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 116 XM_008657227.2 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 116 XM_020542336.1 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 116 NM_001305945.1 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 120 NM_001305944.1 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 120 NM_001112406.2 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 120 JF915836.1 TCTCGCGGGTCTTATCGACTCCAACAAGAACACACTACACACCAGCCAGCGAGATAGCGA 120 *****************************************************
AB218832.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 113 XM_023300797.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 176 XM_008657225.2 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 176 XM_008657227.2 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 176 XM_020542336.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 176 NM_001305945.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 180 NM_001305944.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 180 NM_001112406.2 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 180 JF915836.1 ACGCTAGGAACCCAGTGGCCATCTTTGGAGCGGCCATGACGCTGGATCAGAACCATGCCA 180 ************************************************************
AB218832.1 TGCCGATGCAGCCCCCGGCCCTG - 136 XM_023300797.1 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAG - 210 XM_008657225.2 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG 236
Trang 3XM_008657227.2 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG 236 XM_020542336.1 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG 236 NM_001305945.1 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG 240 NM_001305944.1 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAGAGCATATGGAGCAGAGGGCAGTGCTG 240
NM_001112406.2 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAG - 214
JF915836.1 TGCCGATGCAGCCCCCGGCCCTGCAGCCCGGAAG - 214
***********************
AB218832.1 - 136
XM_023300797.1 - 210
XM_008657225.2 TGGTGCATGGTTCCATCAGAACAGTAGCAGGAGGCCCTACTCTTGCCTTGAATGAGTGCC 296 XM_008657227.2 TGGTGCATGGTTCCATCAGAACAGTA -GGAGGCCCTACTCTTGCCTTGAATGAGTGCC 293 XM_020542336.1 TGGTGCATGGTTCCATCAGAACAGTAGCAG - 266
NM_001305945.1 TGGTGCATGGTTCCATCAGAACAGTAGG - 268
NM_001305944.1 TGGTGCATGGTTCCATCAGAACAGTAGCAGGAGGCCCTACTCTTGCCTTGAATGAGTGCC 300 NM_001112406.2 - 214
JF915836.1 - 214
AB218832.1 -CAGCCCGGAA 146 XM_023300797.1 -CA 212 XM_008657225.2 AGATTTTGGTACAGCAGAAACCCCAGGGAGATTCACGACTGCTGGCCTCTAATCTGTGCA 356 XM_008657227.2 AGATTTTGGTACAGCAGAAACCCCAGGGAGATTCACGACTGCTGGCCTCTAATCTGTGCA 353 XM_020542336.1 - 266
NM_001305945.1 -A 269 NM_001305944.1 AGATTTTGGTACAGCAGAAACCCCAGGGAGATTCACGACTGCTGGCCTCTAATCTGTGGA 360 NM_001112406.2 -GA 216 JF915836.1 -GA 216
AB218832.1 GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 206 XM_023300797.1 GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 272 XM_008657225.2 GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 416 XM_008657227.2 GGAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 413 XM_020542336.1 -GAAGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 325 NM_001305945.1 -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 326 NM_001305944.1 -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 417 NM_001112406.2 -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 273 JF915836.1 -AGAAGCGACCTCGCAGATCACGAGATGGGCCTACGTCAGTGGCAGCTGTCATCCAGC 273 *********************************************************
AB218832.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 266 XM_023300797.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 332 XM_008657225.2 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 476 XM_008657227.2 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 473 XM_020542336.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 385 NM_001305945.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 386 NM_001305944.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 477 NM_001112406.2 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 333 JF915836.1 GGTGGGCTGAGCGCAACAAGCATTTGGAGTATGAGGAATCTGAGGAGGCAAAGCGACCAA 333 ************************************************************
AB218832.1 GAAAAGCACCTGCAAAGGGTTCGAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 326 XM_023300797.1 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 392 XM_008657225.2 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 536 XM_008657227.2 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 533 XM_020542336.1 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 445 NM_001305945.1 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 446 NM_001305944.1 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 537 NM_001112406.2 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 393 JF915836.1 GAAAAGCACCTGCAAAGGGTTCAAAGAAGGGCTGTATGAAGGGAAAAGGGGGGCCTGACA 393 ********************** *************************************
AB218832.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 386 XM_023300797.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 452 XM_008657225.2 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 596 XM_008657227.2 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 593 XM_020542336.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 505 NM_001305945.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 506 NM_001305944.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 597 NM_001112406.2 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 453 JF915836.1 ATACTCAATGTGGATACCGTGGAGTGAGGCAGCGTACTTGGGGGAAGTGGGTTGCTGAAA 453 ************************************************************
AB218832.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 446 XM_023300797.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 512 XM_008657225.2 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 656 XM_008657227.2 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 653 XM_020542336.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 565 NM_001305945.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 566
Trang 4NM_001305944.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 657 NM_001112406.2 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 513 JF915836.1 TAAGAGAGCCAAATCGTGTCGACAGACTCTGGCTGGGTACCTTCCCAACCGCGGAGGATG 513 ************************************************************
AB218832.1 CAGCTAGGGCCTATGACGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 506 XM_023300797.1 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 572 XM_008657225.2 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 716 XM_008657227.2 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 713 XM_020542336.1 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 625 NM_001305945.1 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 626 NM_001305944.1 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 717 NM_001112406.2 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 573 JF915836.1 CAGCTAGGGCCTATGATGAGGCAGCCAGAGCGATGTATGGAGACTTGGCACGGACTAACT 573 **************** *******************************************
AB218832.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTAGCATCGACCTCTGCCCAGG 566 XM_023300797.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 632 XM_008657225.2 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 776 XM_008657227.2 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 773 XM_020542336.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 685 NM_001305945.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 686 NM_001305944.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 777 NM_001112406.2 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 633 JF915836.1 TCCCCGGACAGGATGCAACAACCTCTGCCCAAGCTGCTCTATCATCGACCTCTGCCCAGG 633 ***************************************** ******************
AB218832.1 CTGCTCCAACAGCTGTCGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 626 XM_023300797.1 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 692 XM_008657225.2 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 836 XM_008657227.2 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 833 XM_020542336.1 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 745 NM_001305945.1 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 746 NM_001305944.1 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 837 NM_001112406.2 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 693 JF915836.1 CTGCTCCAACAGCTGTTGAAGCTCTTCAGACTGGCACGTCATGCGAGTCGACAACGACAT 693 **************** *******************************************
AB218832.1 CAAATTACTCGGACATCGCATCCACCTCACACAAGCCTGAAGCCTCTGACATCTCGAGCT 686 XM_023300797.1 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 752 XM_008657225.2 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 896 XM_008657227.2 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 893 XM_020542336.1 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 805 NM_001305945.1 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 806 NM_001305944.1 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 897 NM_001112406.2 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 753 JF915836.1 CAAATCACTCGGACATCGCATCCACCTCACACAAGCTTGAAGCCTCTGACATCTCGAGCT 753 ***** ****************************** ***********************
AB218832.1 CCCTAAAGGCAAAATGCCCAGCTGGATCATGTGGTATCCAAGAGGGTACACCCAGTGTAG 746 XM_023300797.1 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 812 XM_008657225.2 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 956 XM_008657227.2 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 953 XM_020542336.1 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 865 NM_001305945.1 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 866 NM_001305944.1 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 957 NM_001112406.2 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 813 JF915836.1 ACCTAAAGGAAAAATGTCCAGCTGGATCATGTGGTATCCAAGACGGCACACCCATTGTAG 813 ******** ****** ************************** ** ******* *****
AB218832.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 806 XM_023300797.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 872 XM_008657225.2 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 1016 XM_008657227.2 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 1013 XM_020542336.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 925 NM_001305945.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 926 NM_001305944.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 1017 NM_001112406.2 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 873 JF915836.1 CTGACAAGGAGGTCTTTGGGCCGTTGGAGCCTATCACAAATCTTCCAGATGGTGGTGATG 873 ************************************************************
AB218832.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 866 XM_023300797.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 932 XM_008657225.2 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 1076 XM_008657227.2 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 1073 XM_020542336.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 985 NM_001305945.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 986 NM_001305944.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 1077 NM_001112406.2 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 933 JF915836.1 GTTTTGATATCGGTGAGATGCTGAGGATGATGGAAAGCGATCCACATAATGCAGGTGGAG 933
Trang 5************************************************************
AB218832.1 CTGACGCTGGCATGGGGCAGCCCTGGTGTCTTGATGAGCTGGATTCGAGTGTCTTGGAGA 926 XM_023300797.1 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 992 XM_008657225.2 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 1136 XM_008657227.2 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 1133 XM_020542336.1 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 1045 NM_001305945.1 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 1046 NM_001305944.1 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 1137 NM_001112406.2 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 993 JF915836.1 CCGACGCTGGCATGGGACAGCCCTGGTATCTTGATGAGCTGGATTCGAGTGTCTTAGAGA 993
* ************** ********** *************************** ****
AB218832.1 GCATGCTCCAGCCACAGC -CAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 980 XM_023300797.1 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1052 XM_008657225.2 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1196 XM_008657227.2 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1193 XM_020542336.1 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1105 NM_001305945.1 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1106 NM_001305944.1 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1197 NM_001112406.2 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1053 JF915836.1 GCATGCTCCAGCCGGAGCCGGAGCCAGAGCCAGAGCCATTCCTGATGTCTGAAGAACCGG 1053 ************* *** ************************************
AB218832.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1040 XM_023300797.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1112 XM_008657225.2 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1256 XM_008657227.2 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1253 XM_020542336.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1165 NM_001305945.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1166 NM_001305944.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1257 NM_001112406.2 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1113 JF915836.1 ACATGTTTCTTGCTGGCTTCGAAAGCGCTGGTTTCGTCGAGGGTCTGGAGCGGCTAAACT 1113 ************************************************************
AB218832.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1100 XM_023300797.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1172 XM_008657225.2 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1316 XM_008657227.2 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1313 XM_020542336.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1225 NM_001305945.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1226 NM_001305944.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1317 NM_001112406.2 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1173 JF915836.1 GAATTTCTGATGTTTGACCGTTGATCCTCATCCCACTTCATGTCTGAGCTTGTGAATTCG 1173 ************************************************************
AB218832.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTTATATTCCTCTGT 1160 XM_023300797.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1230 XM_008657225.2 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1374 XM_008657227.2 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1371 XM_020542336.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1283 NM_001305945.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1284 NM_001305944.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1375 NM_001112406.2 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1231 JF915836.1 GAGGCAAACATTGGCAGAACTTATAAGCTCTAGCAATTCTAGGCTTTACATTCCT CTG 1231 *********************************************** * *
AB218832.1 AAATAGTTCTCTAGTCATGGGAACTGGGTTTGCTTCACATTTTTTGTAAGACCAGAAGTG 1220 XM_023300797.1 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1290 XM_008657225.2 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1434 XM_008657227.2 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1431 XM_020542336.1 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1343 NM_001305945.1 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1344 NM_001305944.1 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1435 NM_001112406.2 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1291 JF915836.1 TGATAGTTCTCTATGCATGGGAACTGGGTTTGCTTCACAATTTTTGTAAGACCAGAAGTG 1291 *********** ************************ ********************
AB218832.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1280 XM_023300797.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1350 XM_008657225.2 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1494 XM_008657227.2 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1491 XM_020542336.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1403 NM_001305945.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1404 NM_001305944.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1495 NM_001112406.2 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1351 JF915836.1 ATGTAAATAGTTCCCACCTTGTGGAAGGACAAGAAAAAAATAAATAAAAAGAGTGCTTGC 1351 ************************************************************
AB218832.1 TTT - 1283
Trang 6XM_023300797.1 TTTCGTTCCTTA 1362
XM_008657225.2 TTTCGTTCCTTA 1506
XM_008657227.2 TTTCGTTCCTTA 1503
XM_020542336.1 TTTCGTTCCTTA 1415
NM_001305945.1 TTTCGTTCCTTA 1416
NM_001305944.1 TTTCGTTCCTTA 1507
NM_001112406.2 TTTCGTTCCTTA 1363
JF915836.1 TTTC - 1355
***
Từ kết quả, nhận thấy gene NM_001305944.1 (1507 bp) chứa nhiều trình tự bảo thủ, độ dài lớn, là một gene ngô ở Trung Quốc, nên có khả năng sẽ tương đồng với gene ngô ở Việt Nam cần nghiên cứu
Đoạn bảo thủ: (nhiều dấu sao): Vị trí 68-203 và 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:
Trang 7Từ kết quả lựa chọn mồi Primer Pair 4có: mồi xuôi gần đầu trình tự gene gốc và mồi ngược gần cuối gene gốc, có tỉ lệ Self complementarity tương đối thấp, nhiệt độ bắt cặp thích hợp, tỉ lệ GC% nằm trong mức tối ưu…
Trang 83 Kiểm tra mồi:
• Kết quả sau khi chạy PCR ảo trên 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 quả trên có kích thước tương đương với đoạn gene định nhân lên
• So sánh tương đồng của mạch ảo so với các chuỗi tương ứng lưu trữ
trong ngân hàng gen bằng Clustal Omega:
Trang 10- Tương tự khi so sánh trình tự PCR test với các gene khác cũng cho thấy
sự tương đồng lớn
=> Đoạn bảo thủ được lựa chọn tương đối phù hợp
• Đối chiếu lên Blast: https://blast.ncbi.nlm.nih.gov/Blast.cgi
Chọn Nucleotide => Protein Thu được kết quả:
Protein tương đồng cao nhất với mục tiêu:
>NP_001292873.1 dehydration-responsive element binding protein 2A isoform 1 [Zea mays]
MTLDQNHAMPMQPPALQPGRAYGAEGSAVVHGSIRTVAGGPTLALNECQILVQQKPQGDSRLLASNLWKK
RPRRSRDGPTSVAAVIQRWAERNKHLEYEESEEAKRPRKAPAKGSKKGCMKGKGGPDNTQCGYRGVRQRT
WGKWVAEIREPNRVDRLWLGTFPTAEDAARAYDEAARAMYGDLARTNFPGQDATTSAQAALSSTSAQAAP
TAVEALQTGTSCESTTTSNHSDIASTSHKLEASDISSYLKEKCPAGSCGIQDGTPIVADKEVFGPLEPIT
NLPDGGDGFDIGEMLRMMESDPHNAGGADAGMGQPWYLDELDSSVLESMLQPEPEPEPEPFLMSEEPDMF
LAGFESAGFVEGLERLN