Phân lập đoạn gen cp từ soybean mosaic virus và phát triển vector chuyển gen mang cấu trúc rnai phục vụ tạo cây chuyển gen kháng bệnh

BỘ GIÁO DỤC VÀ ĐÀO TẠO ĐẠI HỌC THÁI NGUYấN Lề TH MAI THU PHÂN LậP ĐOạN GEN CP Từ SOYBEAN MOSAIC VIRUS Và PHáT TRIểN VECTOR CHUYểN GEN MANG CấU TRúC RNAi PHụC Vụ TạO CÂY ĐậU TƯƠNG CHUN GEN KH¸NG BƯNH Chun ngành: Di truyền học Mã số: 62 42 01 21 LUẬN ÁN TIẾN SĨ SINH HỌC Người hướng dẫn khoa học: GS.TS Chu Hoàng Mậu PGS.TS Chu Hoàng Hà THÁI NGUYÊN - 2014 Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ i LỜI CAM ĐOAN Tơi xin cam đoan cơng trình nghiên cứu tơi Các kết trình bày luận án trung thực, phần đƣợc công bố Tạp chí khoa học với đồng ý cho phép đồng tác giả, phần cịn lại chƣa đƣợc cơng bố cơng trình khác Tác giả Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ ii LỜI CẢM ƠN Tôi xin bày tỏ lịng biết ơn tới GS.TS Chu Hồng Mậu, PGS.TS Chu Hồng Hà tận tình hƣớng dẫn, tạo điều kiện thuận lợi cho tơi suốt q trình học tập, nghiên cứu hồn thành luận án Tơi xin cảm ơn Phịng Thí nghiệm trọng điểm Cơng nghệ gen, cảm ơn TS Lê Văn Sơn toàn thể cán , Phịng Cơng nghệ tế bào thực vật thuộc Viện Cơng nghệ Sinh học tận tình giúp đỡ, truyền đạt kinh nghiệm quý báu cho trình nghiên cứu thực đề tài luận án Xin cảm ơn giúp đỡ ThS , ThS Tôi xin chân thành cảm ơn PGS.TS Nguyễn Thị Tâm tập thể cán Bộ môn Di truyền & Sinh học đại giúp đỡ, tạo điều kiện thuận lợi cho tơi suốt q trình học tập nghiên cứu đề tài luận án Tôi xin cảm ơn thầy cô Khoa Sinh-Kỹ thuật nông nghiệp Bộ phận quản lý nghiên cứu sinh, Phòng đào tạo, trƣờng Đại học Sƣ phạm – Đại học Thái Nguyên, xin cảm ơn Lãnh đạo trƣờng Đại học Sƣ phạm Lãnh đạo Đại học Thái Nguyên Tôi xin cảm ơn giúp đỡ tạo điều kiện Ban chủ nhiệm khoa Sinh Tôi xin cảm ơn gia đình bạn bè ln động viên, giúp đỡ tơi suốt q trình học tập thực thành công luận án Nghiên cứu sinh Mai Thu Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ iii MỤC LỤC Trang Lời cam đoan i Lời cảm ơn ii Mục lục iii vi viii x MỞ ĐẦU 1 Đặt vấn đề ………………………………………………… ……… Mục tiêu nghiên cứu……………………………………… .……… Nội dung nghiên cứu……………………………………… ……… Những đóng góp luận án………………………… Ý nghĩa khoa học thực tiễn…………………………… ………… Chƣơng TỔNG QUAN TÀI LIỆU…………………… 1.1 BỆNH KHẢM DO VIRUS Ở CÂY ĐẬU TƢƠNG……… .…… ệnh virus đậu tƣơng……………… …… 1.1.2 Sự xâm nhập SMV BYMV vào tế bào đậu tƣơng… .…… 1.1.3 Hệ gen SMV BYMV…………………………… .……… 10 1.2 NGHIÊN CỨU CHUYỂN GEN Ở CÂY ĐẬU TƢƠNG 16 1.2.1 16 1.1.1 … 1.2.2 Định hƣớng ứng dụng số thành tựu chuyển gen đậu tƣơng……………………………… …………………………… Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ 23 iv 1.2.3 Tình hình nghiên cứu tạo đậu tƣơng biến đổi gen Việt Nam… 28 1.3 KỸ THUẬT RNAi TRONG TẠO CÂY CHUYỂN GEN KHÁNG VIRUS………………………………………………………… …………… 29 1.3.1 Các chế RNAi ức chế gen thực vật…………… ………… 30 1.3.2 Ứng dụng kỹ thuật RNAi nghiên cứu tạo chuyển gen kháng virus………………………………………………………………… 36 Chƣơng VẬT LIỆU VÀ PHƢƠNG PHÁP NGHIÊN CỨU .… 40 2.1 VẬT LIỆU, THIẾT BỊ, HÓA CHẤT VÀ ĐỊA ĐIỂM NGHIÊN CỨU 40 2.1.1 Vật liệu thực vật…………………………………… ………… 40 2.1.2 Vector chủng vi khuẩn ……………………… .…………… 42 2.1.3 Hóa chất thiết bị ……………………………… …………… 42 2.1.4 Địa điểm nghiên cứu……………………………… .…………… 42 2.2 PHƢƠNG PHÁP NGHIÊN CỨU……………………… .……… 43 2.2.1 Nhóm phƣơng pháp phân lập gen………………… 43 2.2.2 Nhóm phƣơng pháp thiết kế vector chuyển gen mang cấu trúc RNAi 48 2.2.3 Phƣơng pháp chuyển gen thông qua A tumefaciens 52 2.3.4 Nhóm phƣơng pháp phân tích chuyển gen 58 Chƣơng KẾT QUẢ NGHIÊN CỨU VÀ THẢO LUẬN… … 60 3.1 TÁCH DÒNG VÀ PHÂN TÍCH TRÌNH TỰ ĐOẠN GEN CP CỦA SMV……………………………………………… …… 60 3.1.1 Tách dòng xác định trình tự nucleotide đoạn gen CP từ SMV.…… 60 3.1.2 Phân tích đa dạng trình tự gen CP dịng SMV 67 Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ v 3.2 THIẾT KẾ VECTOR CHUYỂN GEN MANG CẤU TRÚC RNAi … 74 3.2.1 Thiết kế vector chuyển gen mang cấu trúc RNAi kháng đơn loài SMV………………………………………………………… …………… 74 3.2.2 Thiết kế vector chuyển gen mang cấu trúc RNAi kháng hai loài SMV BYMV…………………………………………………………………… 81 3.3 KẾT QUẢ TẠO CÂY THUỐC LÁ CHUYỂN GEN KHÁNG SMV VÀ BYMV………………………………………………… ………… 89 3.3.1 Kết chuyển cấu trúc CPi (SMV-BYMV) vào thuốc lá… 89 ……… .……………… 92 RNAi … 96 … 96 3.4 K ut dòng đậu - 0………… 99 KẾT LUẬN VÀ ĐỀ NGHỊ…………………… .…………… 106 KẾT LUẬN…………………………………………… …………… 106 ĐỀ NGHỊ……………………………………………… .…………… 107 108 TÀI LIỆU THAM KHẢO…………………………… 109 Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ vi DANH MỤC CHỮ VIẾT TẮT A tumefaciens Agrobacterium tumefaciens AS Acetosyrigone BAP 6-benzyladenine purin BGM Bean golden mosaic virus bp Base pair BYMV Bean yellow mosaic virus CCM Co-cultivation medium CAMV Cauliflower mosaic virus CMV Cucumber mosaic virus ) CP ) ) CPi –SMV Đoạn bảo thủ đƣợc thiết kế từ gen CP SMV CPi(SMV-BYMV) Đoạn bảo thủ đƣợc thiết kế từ gen CP SMV BYMV DNA Deoxyribo nucleic acid dNTP Deoxynucleoside triphosphate đtg Đồng tác giả E coli Escherichia coli EDTA Ethylene diamine tetraacetic acid GA3 Gibberellic acid GM Germination medium (mơi trƣờng nảy mầm) gus Gen mã hóa enzyme β-Glucuronidase IAA Indoleacetic acid IBA Indole-3-butyric acid kb Kilo base LB Luria and Bertani Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ vii MS Murashige & Skoog (1962) -Môi trƣờng NAA α-Naphthaleneacetic acid nptII Neomycin phosphotransferase gene OD Optical density – Mật độ quang PCR Polymerase Chain Reaction – Phản ứng chuỗi Polymerase PPT Phosphinothricin PRSV Papaya ringspot virus pK7GW-CPi-SMV: Vector chuyển gen mang cấu trúc CPi-SMV pK7GW-CPi (SMV-BYMV) Vector chuyển gen mang cấu trúc CPi (SMVBYMV) RM Rooting medium (môi trƣờng rễ) RNAi RNA interference SDS Sodium dodecylsulfat SEM Shoot elongation medium (môi trƣờng kéo dài chồi) SIM Shoot induction medium (môi trƣờng tạo chồi) SL1 SMV dòng Sơn La - Việt Nam SL2 SMV dòng Sơn La - Việt Nam SMV Soybean mosaic virus Taq Thermus aquaticus T-DNA Vùng DNA plasmid chuyển vào thực vật Ti- plasmid Plasmid tạo khối u TMV Tobacco mosaic virus TYLCV Tomato yellow leaf curl virus v/p vòng/phút Vir Virulence Region X-gluc 5-bromo-4-chloro-3-indolyl glucuronide YEP Yeast extract peptone Số hóa Trung tâm Học liệu ) ) http://www.lrc-tnu.edu.vn/ viii DANH MỤC BẢNG Trang Bảng 1.1 2014 Bảng 2.1 Thành phần phản ứng tổng hợp cDNA 45 Bảng 2.2 Thành phần phản ứng PCR 46 Bảng 2.3 Thành phần phản ứng ghép nối gen vào vector pBT…… 47 Bảng 2.4 Thành phần phản ứng ghép nối gen vào vector pENTRTM/D-TOPO…………………………………… 50 Bảng 2.5 Thành phần phản ứng LR……………………………… 51 Bảng 2.6 Thành phần phản ứng cắt enzyme hạn chế……… 52 Bảng 2.7 Thành phần loại môi trƣờng tái sinh in vitro……… 53 Bảng 2.8 Thành phần dung dịch đệm tách DNA tổng số………… 58 Bảng 3.1 Trình tự cặp mồi PCR thiết kế sử dụng nhân đoạn gen CP…………………………………………………… 60 Bảng 3.2 Các vị trí sai khác trình tự đoạn gen CP SMV dịng SL2 so với dịng SL1 trình tự có mã số X63771 65 Bảng 3.3 Các vị trí sai khác trình tự amino acid protein suy diễn dịng SL2 so với dịng SL1 trình tự có mã số CAA45307……………………………………………… 66 Bảng 3.4 Ngân hàng gen quốc tế ng phân tích ………………………… Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ 68 ix Bảng 3.5 ………………… 69 Bảng 3.6 Trình tự cặp mồi SMV-CPi-Fi/SMV-CPi-Ri …………… 76 Bảng 3.7 Trình tự cặp mồi SMV-CPi-Fi/BYMV-CPi-Ri ………… 83 Bảng 3.8 Kết biến nạp cấu trúc CPi (SMV-BYMV) vào mảnh thuốc C9-1………………………………………… 91 gen………………………… 95 Bảng 3.9 Bảng 3.10 Kết biến nạp cấu trúc 2008 …………………… Số hóa Trung tâm Học liệu http://www.lrc-tnu.edu.vn/ 99 112 28 Afanasiev M M., Morris H E (1952), “Bean Virus (yellow) on Great Northern bean in Montana”, Phytopathology, 42, pp 101-104 29 Ambros V., Bartel B., Bartel D P., Burge, C B., Carrington J C., Chen X., Dreyfuss G., Eddy S R., Griffiths J.S., Marshall M (2003), “A uniform system for microRNA annotation”, RNA 9, pp 277 - 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RNAi, (2) Biến nạp vector chuyển gen mang cấu trúc RNAi vào thông qua vi khuẩn A tumefaciens làm bất hoạt mRNA virus gây bệnh, (3) Sàng lọc chuyển gen mang cấu trúc RNAi kiểm tra tính kháng virus