Chọn lọc sau biến nạp gen AtZAT12 trên cây Arabidopsis

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Tác giả: Thị Tuyết Châm Lê, Thị Ngọc Khoa Phạm

Ngôn ngữ: Vie

Ký hiệu phân loại: 576.5 Genetics

Thông tin xuất bản: Tạp chí Nông nghiệp và Phát triển, 2023

Mô tả vật lý: 32-39

Bộ sưu tập: Metadata

ID: 422352

Thêm vào giỏ Liên kết toàn văn

Nghiên cứu này nhằm chọn lọc các hạt Arabidopsis sau chuyển gen AtZAT12 gián tiếp qua Agrobacterium tumerfaciens. ZAT12 là một yếu tố phiên mã có chức năng ức chế yếu tố phiên mã khác là FIT thông qua motif EAR. Bản thân FIT cũng là một yếu tố phiên mã trung tâm điều khiển quá trình hấp thụ Fe trong điều kiện môi trường thiếu Fe. Tuy nhiên, khi Fe được hấp thụ quá nhiều sẽ sản sinh các gốc tự do gây tổn hại tế bào. Có lẽ đó là lý do phiên mã AtZAT12 được tăng cao còn phiên mã của FIT bị ức chế trong điều kiện thiếu Fe kéo dài 10 ngày. Để nghiên cứu chức năng, gen AtZAT12 đã được gắn vào vector pMDC107 để chuyển vào cây Arabidopsis. Hạt Arabidopsis T0 thu được sau quá trình biến nạp bằng phương pháp nhúng hoa được đặt trên môi trường thạch MS 1% Agar bổ sung Hygromycin B 15 μg/mL. Cây Arabidopsis con kháng hygromycin B có các lá mầm dài (~ 1,0 cm), trong khi các cây con không kháng kháng sinh có các lá mầm ngắn (~ 0,3 cm). Phương pháp chọn lọc cải tiến rút ngắn thời gian xác định cây Arabidopsis con biến đổi gen nhanh hơn chỉ trong 3,25 ngày. Sau đó, các cây được chọn sẽ được kiểm tra sự có mặt của gen bằng PCR cũng như sự hoạt động của gen chuyển AtZAT12 dưới kính hiển vi., Tóm tắt tiếng anh, This study aimed to select Arabidopsis seeds after transformation with the AtZAT12 gene via Agrobacterium tumerfaciens. ZAT12 is a transcription factor that inhibits other transcription factors FIT through the EAR motif. FIT itself is a central transcription factor that controls Fe uptake under Fe-deficient conditions. However, if Fe is absorbed excessively, it will produce reactive oxygen species that could damage cells. That is the reason why AtZAT12 transcription is elevated and FIT transcription is inhibited under Fe deficiency for 10 days. To investigate the function, the AtZAT12 gene was inserted into the pMDC107 vector for transformation into Arabidopsis. The T0 Arabidopsis seeds obtained after floral dip transformation were placed on 1% MS agar supplemented with 15 μg/mL Hygromycin B. Beforehand, the T0 seeds were sterilized and kept in the dark for stratification. Subsequently, the seed plates were subjected to a regime of 6 h of light, 48 h of dark and 24 h of light (3.25 d). The hygromycin B-resistant seedlings had long hypocotyls (~ 1.0 cm), while the non-resistant seedlings had short (~ 0.3 cm) hypocotyls. This method took only 3.25 days to identify transgenic Arabidopsis seedlings. After that, positive transgene lines were examined by PCR method for AtZAT12, and the expression of AtZAT12 was observed under microscope.

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