Luận án Nghiên cứu thiết kế và chuyển gen agpopt tổng hợp nhân tạo vào cây sắn (manihot esculenta crantz)

Cuối cùng chúng tôi tiến hành phân tích trình tự nhận biết của enzim cắt giới hạn và các thành tố của trình tự gen tổng hợp bằng phần mềm SnapGene Viewer. Các vị trí nhận diện duy nhất của enzim cắt giới hạn và các trình tự thông dụng của quá trình tạo dòng được thể hiện trên bản đồ tượng trưng của gen. Theo như phân tích, hai enzim XbaI và SacI có trình tự nhận diện duy nhất được bổ sung tương ứng ở đầu 5‘ phosphate và 3‘OH. Ngoài ra, một trình tự mã hóa cho đuôi C-Myc/KDEL cùng khung đọc (frame) tiếp nối sau codon mã hóa cuối cùng của gen. Tóm lại, gen AGPopt đã được tổng hợp cho bước tiếp theo tạo dòng vào vector biểu hiện.

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es it was 82% in KM94 compared with 59.6% in KM140. On somatic embryo germination medium containing 0.3 mg/L BAP, there were 71 - 89% somatic embryo masses of KM140 and 79 – 79.2% of KM94 turned up into healthy plants. The regenerated plants produced strong roots (3-4 roots/shoot) on the MS medium without growth hormone. To develop the genetic transformation procedure for studied cassava varieties, Gus gene (in pBI121 vector) was transformed into KM140 using A. tumefaciens method. PCR using nptII_F/R primers indicated that 17 regenerated plants were putative transgenic plants. The gene transformation efficiency was 0.9%. Further modification was carried out in order to have more available initial explants for transformation. Green, hard and shiny cotyledons formed from 115 primary somatic embryos were separated, cut into 4 mm 2 pieces and inoculated with bacterial suspension for 15 minutes. The secondry cotyledon pieces were placed in co-culture medium CIM (MS + picloram 12 mg/L) containing acetosynringone at 100 mM concentration. After two days of co-culture, transgenic cotyledon pieces were washed with cefotacim antibiotics at concentration of 500 mg/L to remove extra A. tumefaciens. Then, they were transferred to a new CIM medium containing 500 mg/L cefotacim and 50 mg/L kanamycin (to select nptII). This modification in the transformation procedure obvious enhanced the performance/effectiveness of gene transfer to studied cassava varieties. Putative transgenic plant analysis using gus assay and PCR with gene specific primers indicated a high number of plant carring transferred genes.. Based on the data of the glgC protein of E. coli K12 (code number AKD93525.1) and the mutation G336D, a DNA sequence encoding an enzyme of 431 amino acid was designed. After optimizing codon usage for gene effective expressed in plants, DNA sequence was linked with a nucleotide segment encoding a signal peptide of rbcS protein (Arabidopsis) for transporting proteins into chloroplasts in the 5‘end and a segment of DNA coding for c-myc and KDEL tail at 3‘ end. Two recognition positions of restriction enzymes XbaI and SacI were added to the 5 'and 3' end of the gene in the respective order. Finally, the designed AGPopt gene encoding AGPase of 1527 bps long, was synthesized by Integrated DNA Technologies (USA) agent and cloned in the vector pUCIDT-AMP. Then AGPopt genes were inserted to pBI121 plant expression vector in and transferred in to A. tumefaciens CV58/pGV2260 for transformation, The constructed pBI121 vector harboring AGPopt gene has been tested using model tobacco plant. PCR using the primers G336F/G336KDEL R proved 32 out of 39 regenerated lines were positive; Southern blot resulted that all 8 tested lines shown AGPopt copies in their genomic. Further analysis of AGPase activity in the transgenic tobacco lines indicated the increase from 121% (L6) to 153% (L7) and 116 the starch contents also increased from 140.05% (L2) to 168.99% (L7) compared to the non-transgenic control. The transformation of AGPopt vector into cassava was conducted with 1018 explants in 6 experimental plots. Totally, there were 69 regenerated plants produced (6.78%), however only 6 plants form heathy roots. PCR analysis for the presence of transgene structure using nptII_F / R primers proved 5 out of 6 lines were positive. By using specific primers G336 F/G336KDEL R , PCR bands have been found at the size of 1.5 kb, corresponding to AGPopt gene. Following, double PCR concluded that 5 regenerated cassava lines are possible transgenic plants. Further experiments to prove the transgenic cassava lines are going on. Results of this study are leading to following conclusions: 1. In vitro tissue culture and plant regeneration via somatic embryogenesis using shoot apex and young leaves have been optimized for two local cassava varieties KM140 and KM94, suitable for genetic transformation. Somatic embryo was produced on MS medium supplied with 12 mg/lpicloram and germinated with the present of 0,3 mg/l BAP. 2. The genetic transformation procedure for studied cultivars was optimized using GUS gene with the efficiency of 0.9%. 3. Based on amino acid sequence of GlgC protein (Accession number AKD93525.1) of E. coli K12, an AGPopt gene has been synthesized, in which codon usage has been optimized for efficient expression of AGP in plant and glycine at position 336 was substituted by aspartate (G336D) for enhancing APG activity. AGPopt gene was then inserted into the plant expression vector pBI121 and this recombination vector was transferred into tobacco for evaluation before introducing into target species. 4. AGPopt gene was successfully transferred to cassava cultivar KM94 and transgenic plants were regenerated through somatic embryo development. Primarily, PCR amplification had proved there were 5 out of 6 obtained cassava plants containing nptII and AGPopt genes in their genomes. 117 TÀI LIỆU THAM KHẢO 1. Abhary M, Siritunga D, Stevens G, Taylor NJ, Fauquet CM (2011) Transgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta) Generates a Novel Sink for Protein. PLoS ONE 6(1). 2. Abraham TE (1996) Dry extraction of starch from cassava tubers. In: Kurup GT, Palaniswami MS, Pottty VP, Padmaja G, Kabeerathumma S, Pillai SV, eds. Tropical Tuber Crops: problems, prospects and future strategies. Science Publisher INC., Lebanon, New Hampshire. 3. 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Chủng vi khuẩn/vector A. tumefaciens CV58/pGV2260/pBI121 2. Phương pháp và vật liệu lây nhiễm Lắc mẫu với huyền phù vi khuẩn với tốc độ 50 v/p; mảnh lá mầm 3. Thời điểm lây nhiễm và mật độ vi khuẩn Mật độ khuẩn tại OD 600 0,8 Thời điểm: sau khi cảm ứng trên môi trường tạo phôi soma 2 ngày 4. Nồng độ AS và thời gian lây nhiễm AS 100 μM; Thời gian lây nhiễm 15 phút 5. Thời gian đồng nuôi cấy thích hợp AS 150 μM; Thời gian đồng nuôi cấy 2 ngày 6. Loại kháng sinh và ngưỡng chọn lọc thích hợp Kanamycin 50 mg/l 7 8. Hoạt tính AGPase ở lá cây thuốc lá K326/AGPopt Hoạt tính AGPase Tỉ lệ % so với đối chứng ĐC 0.1 100% L1 0.137 137% L2 0.145 145% L3 0.137 137% L4 0.144 144% L5 0.140 140% L6 0.121 121% L7 0.153 153% L8 0.146 146% 9. Thành phần các môi trường nuôi cấy in vitro cơ bản Loại môi trường Thànhh phần MS lỏng MS (I-V) + Sucrose 30 mg/l; pH 5,8 MS đặc MS lỏng + 7,5 g Agar CIM MS (I - V) bổ sung picloram 12 mg/l, sucrose 30 g/l và 7,5g agar, pH 5,8 CEM MS (I - V) bổ sung BA 0,3 mg l, sucrose 30 mg l và 7,5g agar, pH 5,8 10. Thành phần các môi trường nuôi khuẩn Môi trường Thành phần YEB đặc 1 g/l yeast extract + 5 g/l beef extract + 5 g/l NaCl + 5 g/l tryptone + 5 g/l sucrose 15 g/l bacto agar, pH = 7,0 YEB lỏng 1 g/l yeast extract + 5 g/l beef extract + 5 g/l NaCl + 5 g/l tryptone + 5 g/l sucrose, pH = 7,0 LB lỏng 10 g/l Tryptone+ 5 g/l yeast Extract + 10 g/l NaCl, pH = 7,0 LB đặc 10 g/l Tryptone+ 5 g/l yeast Extract + 10 g/l NaCl, 15 g/l bacto agar, pH = 7,0 8 11. Thành phần sol I, sol II, sol III Sol I: 50 mM glucose, 10 mM EDTA, 25 mM Tris (pH 8,0) Sol II: 0,2 N NaOH, 1 % SDS Sol III: 3 M KOAc (pH 6,0)

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