- 學位論文
透過基因編輯技術降低水稻吸收砷和鎘轉運蛋白之表達
Attenuation of Arsenic and Cadmium Uptake Transporter Expression in Rice Through Genome Editing Technology
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摘要
砷和鎘會透過必需元素或有益元素通道進入水稻穀粒，因此如何減少水稻中砷和鎘的累積是農業上的一大挑戰。水稻根部中，矽轉運蛋白OsLsi2 ( Low Silicon transporter 2，簡稱 OsLsi2 )和磷轉運蛋白OsPT4 ( Phosphate transporter 4，簡稱 OsPT4)分別是水稻根部吸收三價砷 (AsⅢ) 和五價砷 (AsⅤ) 主要通道；而錳轉運蛋白OsNRAMP5 ( Natural Resistance-Associated Macrophage Protein 5 ，簡稱OsNRAMP5) 則是吸收鎘的主要管道。為了抑制水稻對砷和鎘的吸收，本試驗分為兩個部份進行：(1)利用CRISPR/Cas9基因編輯技術，同時對OsPT4和OsNRAMP5兩個基因進行精準突變；(2)從實驗室先前藉由對OsLsi2啟動子進行多重編輯，篩選出多種OsLsi2不同表達程度的水稻進行水耕和土耕的砷吸收試驗，以篩選出三價砷吸收降低，且不影響水稻產量之水稻品系。第一部分試驗中利用tRNA串聯技術同時對 OsPT4和OsNRAMP5基因進行敲除，轉殖後共獲得30個T0世代品系，經定序後在其中2個品系觀察到鹼基剔除，並從其T1世代篩選出了6種不同基因型的osnramp5-ospt4雙重突變體；第二部分幼苗試驗結果顯示，29-28品系不論是砷處理或者砷+矽共處理的條件下，皆能使地上部砷的累積量分別下降27.1%和51.9%，其OsLsi2表現量也較WT下降65%，將此品系種植於砷汙染土壤，穀粒中的砷累積量也較WT降低30.9%。未來將進行osnramp5-ospt4雙重突變體的砷和鎘的吸收試驗，並希望能透過上述兩種突變體雜交，創造出可同時降低砷和鎘累積的水稻品系。
並列摘要
Arsenic and cadmium can enter rice grains through essential element or benefit element channels. Therefore, reducing the accumulation of these substances in rice poses a major challenge in agriculture. In rice roots, the silicon transporter OsLsi2 (Low Silicon transporter 2, OsLsi2) serves as the main channel for absorbing trivalent arsenic (AsⅢ), while the phosphorus transporter OsPT4 (Phosphate transporter 4, OsPT4) facilitates the uptake of pentavalent arsenic (AsⅤ). Additionally, the manganese transporter OsNRAMP5 (Natural Resistance-Associated Macrophage Protein 5, OsNRAMP5) is responsible for the absorption of cadmium. This experiment aimed to inhibit the uptake of arsenic and cadmium in rice and was divided into two parts. Firstly, CRISPR/Cas9 gene editing technology was used to precisely mutate the two genes, OsPT4 and OsNRAMP5, simultaneously. Secondly, the OsLsi2 promoter underwent multiple edits to create a variety of rice plants with different expression levels of OsLsi2. These plants were then screened for arsenic uptake in hydroponic and As-contaminated soil. The goal was to identify rice lines that can reduce the uptake of trivaent arsenic without affecting rice yield. In the first part of the experiment, a total of 30 transgenic lines were obtained in the T0 generation, and after sequencing, base deletion was observed in 2 of these lines. In the T1 generation, 6 different genotypes of osnramp5-ospt4 double mutants were screened. In the second part, the line 29-28 showed promising results. It exhibited a reduction of arsenic accumulation in its shoots by 27.1% and 51.9% under arsenic treatment or arsenic+silicon treatment conditions, respectively. When the line was planted in arsenic-contaminated soil, the expression of OsLsi2 was higher compared to the wild type (WT), resulting in a 30.9% reduction in arsenic accumulation in the grain compared to WT. In future experiments, the osnramp5-ospt4 double mutant will undergo arsenic and cadmium absorption tests. The ultimate goal is to crossbreed the above mutants to create a rice line capable of simultaneously reducing the accumulation of both arsenic and cadmium.
並列關鍵字
Rice ； OsLsi2 ； OsPT4 ； OsNRAMP5 ； silicon transporter ； phosphate transporter ； manganese transporter ； CRISPR/Cas9 ； arsenic ； cadmium參考文獻
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