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Đậu nành phốt phát thấp: Kỹ thuật chỉnh sửa gen CRISPR-Cas9 trong công nghệ kỹ thuật chuyển hóa thế hệ tiếp theo
Journal of Plant Biochemistry and Biotechnology - Trang 1-16 - 2023
Tóm tắt
Kỹ thuật chuyển hóa thế hệ tiếp theo mở rộng khả năng sử dụng cây trồng như các nhà máy sinh học để sản xuất hàng loạt các metabolite. Hệ thống CRISPR/Cas9 là công cụ chỉnh sửa gen mới nhất và được áp dụng rộng rãi nhất cho kỹ thuật chuyển hóa nhằm nâng cao tính trạng cây trồng. Trong phương pháp được hướng dẫn bởi RNA này, tất cả các sgRNA không có hiệu quả như nhau và điều quan trọng là phải giảm thiểu rủi ro khi sử dụng sgRNA không hiệu quả tạo ra các biến dị không mong muốn. Trong nghiên cứu này, chúng tôi đã thiết kế hai sgRNA nhắm vào bước cuối cùng của sinh tổng hợp phytat, GmIPK1- exon 6 (KS1) và 1 (KS2) và xác nhận hiệu quả của chúng bằng cách sử dụng các công cụ in silico khác nhau. Chúng tôi đã xác nhận hiệu quả chức năng của sgRNAs bằng cách sử dụng AGRODATE (thí nghiệm đĩa do Agrobacterium thực hiện cho biểu hiện tạm thời) trước khi phát triển các biến thể ổn định. Phân tích thống kê kết hợp về tỷ lệ đột biến trong các biến thể tạm thời cho thấy các đột biến thiếu hụt, cụ thể là KS1_sgRNA1 (76.4%) thuộc khoảng từ 1 đến 7 nucleotide và các biến thể theo chiều cao chiếm (23.4%), trong trường hợp của KS2_sgRNA2, có 85.2% thiếu hụt được quan sát trong khoảng từ 1 đến 6 nucleotide, trong khi các biến thể theo chiều cao chiếm (13.2%). Phân tích trình tự của các sản phẩm khuếch đại cho thấy sự hiện diện của các đột biến trong 12 trong số 16 dòng transgenic dương tính (75%). Phân tích phytat của các biến thể knock-out sgRNA1 cho thấy giảm khoảng 6.6 lần, trong khi biến thể knock-out sgRNA2 cho thấy giảm 7.05 lần trong các biến thể đậu nành T0 ổn định. Chiến lược được thực hiện trong nghiên cứu này là báo cáo đầu tiên về một hệ thống chỉnh sửa CRISPR/Cas9 hiệu quả cao sử dụng gRNA chimeric trong giống đậu nành DS9712, củng cố tầm quan trọng của việc phát triển đậu nành phốt phát thấp với tiềm năng lớn cho ngành thực phẩm và thức ăn chăn nuôi.
Từ khóa
#CRISPR/Cas9 #chỉnh sửa gen #kỹ thuật chuyển hóa #đậu nành phốt phát thấp #sinh tổng hợp phytat #biến thể knock-out.Tài liệu tham khảo
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