Luận án Nghiên cứu tăng cường khả năng chống chịu các điều kiện bất lợi của môi trường trên đối tượng cây Xoan ta (Melia azedarach L.) bằng công nghệ gen thực vật

hơn so với dòng cây đối chứng không chuyển gen sau khi xử lý bởi hạn nhân tạo. 4) Thiết kế đƣợc hai cấu trúc vector chuyển gen mang gen codA dƣới sự điều khiển của promoter 35S (pBI121::TP-codA và pBI121::codA), và chuyển thành công gen TP-codA/codA vào cây thuốc lá thông qua Agrobacterium tumefaciens. Các dòng thuốc lá chuyển gen có sự sinh tổng hợp và tích lũy glycine betaine cao trong lá (1,10 - 6,49 mM/g lá tƣơi) nên tăng cƣờng đƣợc khả năng chịu mặn so với dòng thuốc lá không chuyển gen. Các dòng thuốc lá chuyển cấu trúc gen TP-codA chịu mặn tốt hơn so với các dòng thuốc lá chuyển gen codA. 5) Tạo đƣợc 68 dòng Xoan ta chuyển cấu trúc gen TP-codA. Đánh giá các dòng Xoan ta chuyển gen ở điều kiện hạn và mặn nhân tạo thu đƣợc 5 dòng (TX4, TX12, TX27, TX28 và TX54) chịu hạn và mặn tốt hơn so với dòng cây đối chứng không chuyển gen. Hàm lƣợng glycine betaine tích lũy trong lá của các dòng Xoan ta chuyển gen cao hơn nhiều so với dòng đối chứng không chuyển gen ở điều kiện xử lý bởi hạn và mặn nhân tạo. 136 2. ĐỀ NGHỊ 1) Tiếp tục phân tích, đánh giá khả năng chống chịu các điều kiện lạnh, nhiệt độ cao của các dòng Xoan ta chuyển gen P5CSm, TP-codA ở điều kiện phòng thí nghiệm. Đánh giá khả năng chịu mặn, khô hạn của các dòng Xoan ta chuyển gen trên điều kiện đồng ruộng. 2) Ứng dụng quy trình chuyển gen vào Xoan ta thông qua Agrobacterium tumefaciens xây dựng đƣợc để chuyển các gen mục tiêu có giá trị vào cây Xoan ta tạo giống mới. 3) Sử dụng cấu trúc vector chuyển gen mang gen TP-codA để chuyển vào một số loài cây trồng nông lâm nghiệp khác để sớm tạo ra những giống cây trồng có khả năng chống chịu với điều kiện môi trƣờng bất lợi. 137 NHỮNG CÔNG TRÌNH CÔNG BỐ LIÊN QUAN ĐẾN LUẬN ÁN 1. Bùi Văn Thắng, Phạm Thị Hằng, Đỗ Xuân Đồng, Lê Văn Sơn, Chu Hoàng Hà (2012) Nghiên cứu hoạt động của promoter rd29A cảm ứng hạn ở cây xoan ta (Melia azedarach L.) chuyển gen. Tạp chí KH &CN, VAST 3: 504-510. 2. Bùi Văn Thắng, Đỗ Xuân Đồng, Lê Văn Sơn, Chu Hoàng Hà (2013) Quy trình chuyển gen vào cây xoan ta (Melia azedarach L.) bằng Agrobacterium đạt hiệu suất cao. Tạp chí Sinh học 35(2): 227 - 233. 3. Bùi Văn Thắng, Lê Văn Sơn, Chu Hoàng Hà (2013) Nghiên cứu tạo cây Xoan ta (Melia azedarach L.) chuyển gen P5CSm tăng cƣờng khă năng chống chịu khô hạn. Tạp chí Nông nghiệp & PTNT 1: 203-208. 4. Bùi Văn Thắng, Lê Văn Sơn, Chu Hoàng Hà (2013) Chuyển gen codA mã hóa choline oxidase vào cây Xoan ta (Melia azedarach L.) tăng cƣờng khă năng chịu hạn. Tạp chí Khoa học & Công nghệ Lâm nghiệp 2: 3-10. 5. Bùi Văn Thắng, Lê Văn Sơn, Chu Hoàng Hà (2013) Nghiên cứu tạo cây thuốc lá (Nicotiana tabacum L.) chuyển gen codA mã hóa choline oxidase tăng cƣờng khả năng chịu mặn. Báo cáo khoa học, Hội nghị Khoa học CNSH toàn quốc: 1059- 1063. 6. Đỗ Xuân Đồng, Bùi Văn Thắng, Hồ Văn Giảng, Lê Văn Sơn, Chu Hoàng Hà (2011) Nghiên cứu chuyển gen mã hóa gibberellins 20 –oxidase vào cây Xoan ta (Melia azedarach L.) bằng Agrobacterium tumefaciens. Tạp chí CNSH 9(2): 217- 222. 7. Hồ Văn Giảng, Hà Văn Huân, Vũ Kim Dung, Chu Hoàng Hà, Bùi Văn Thắng (2011). Tạo giống Xoan ta (Melia azedarach L.) sinh trƣởng nhanh bằng kỹ thuật chuyển gen. Tạp chí Nông nghiệp & PTNT: 11-14. 8. Hồ Văn Giảng, Vũ Kim Dung, Hà Văn Huân, Bùi Văn Thắng (2011) Tái sinh cây Xoan ta (Melia azedarach L.) thông qua phôi soma từ rễ cây mầm phục vụ tạo giống cây trồng biến đổi gen. Tạp chí Nông nghiệp & PTNT 2: 206 - 210. 138 SUMMARY 1. Thesis title: “Study on improvement of abiotic stress tolerance in Xoan tree (Melia azedarach L.) by using genetic engineering” 2. Objectives: 2.1. General objectives: Genetic transformation methods were used in order to improve the environmental stress tolerance in Melia azedarach L. Outcomes of this thesis provide scientific evidences for the feasibility of applying modern plant biotechnology to develop novel tree cultivars with improved abiotic stress tolerance. 2.2. Detailed objectives: (1) Establishing and optimizing reliable and reproducible transgenic approaches into Melia azedarach tree; (2) Evaluating efficiency of a dehydration-responsive promoter, rd29A, in transgenic Melia azedarach lines; (3) Evaluating the tolerance to drought stress in transgenic Melia azedarach lines overexpressing a feedback-removed version of P5CS gene; (4) Evaluating the tolerance to drought and salinity stresses in transgenic Melia azedarach and tobacco lines overexpressing codA gene. 3. Contents: (1) Establishing and optimizing reliable and reproducible transgenic approaches into Melia azedarach tree mediated by Agrobacterium tumefacines; (2) Production of transgenic Melia azedarach lines carrying gus-intron gene driven by promoter rd29A (rd29A::gus) into Melia azedarach tree and evaluating the expression level of GUS in transgenic Melia azedarach lines; 139 (3) Transformation of construct carrying P5CSm gene driven by promoter rd29A (rd29A:: P5CSm) into Melia azedarach tree and evaluating the tolerance to drought stress in transgenic Xoan lines based on standard physiological, biochemical aspects; (4) Production of transgenic tobacco and Melia azedarach lines carrying constructs TP-codA and codA driven by promoter 35S, respectively, and evaluating the tolerance to drought and salinity stresses in these transgenic lines based on standard physiological, biochemical aspects. 4. Contributions The thesis is the first evidence in establishing a reliable and reproducible method for genetic transformation mediated by Agrobacterium tumefaciens in Melia azedarach tree. Application of this method, the work successfully produced for the first time transgenic Melia azedarach lines overexpressing P5CSm and codA, respectively, and further proved the potential of tolerance to drought and salinity in vitro of these transgenic lines. In this thesis, a version of bacterial codA gene which the code was optimized for expression in eukaryote cells was overexpressed in tobacco and Melia azedarach tree and especially, analysis of transgenic lines revealed significantly higher accumulation of glycine betaine than that published previously. This thesis provide scientific evidences for the feasibility of applying modern plant biotechnology to develop novel Melia azedarach tree, in particular, and tree cultivars, in general, with improved abiotic stress tolerance. 5. Results: (1) Methods for transformation into Melia azedarach tree mediated by Agrobacterium tumefaciens were successfully established and the transformation efficiency reached to 13,77 – 18,15% in different biological experiments. The methods were proved to be reproducible and reliable for further application in order to genetically modify Melia azedarach tree. 140 (2) Transgenic Melia azedarach lines overexpressing GUS driven by promoter rd29A were successfully produced. Analyses of transgenic Melia azedarach lines confirmed the specific induction of promoter rd29A under drought conditions. (3) 72 transgenic Melia azedarach lines overexpressing a mutated version of P5CS gene (loss of feedback inhibition) driven by promoter rd29A were produced. Analyses of transgenic lines exhibited that two lines PX24 and PX30 showed significantly high tolerance to drought. Proline content accumulated in transgenic lines significantly higher than that observed in control lines under drought conditions. (4) Two transgenic vectors pBI121 carrying codA gene driven by promoter 35S (pBI121::TP-codA và pBI121::codA) were successfully constructed. These vectors were transformed into Agrobacterium tumefaciens strain LAB4404 for further plant transformation. Transgenic tobacco lines overexpressing TP-codA and codA encoding choline oxidase – a key enzyme in glycine betaine biosynthesis, respectively, were successfully produced. Analyses of transgenic tobacco lines observed a tight correlation of the high accumulation (1.10 – 6.49 mM/g fresh leaves) of glycine betaine and the tolerance to salinity. Moreover, it was seen that lines overexpressing TP-codA showed higher tolerance to stress than that of lines overexpressing only codA. 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