Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/635
Title: | 利用單核苷酸多型性資料庫及基因編輯技術建立 OsNramp5 基因突變水稻 Generation of OsNramp5 Dysfunction Rice Mutants Using Single Nucleotide Polymorphism Database and Genome Editing Technology |
Authors: | Yu-hsiang Lin 林煜翔 |
Advisor: | 洪傳揚(Chwan-Yang Hong) |
Keyword: | 鎘,水稻,OsNramp5 基因,單核?酸多態性,CRISPR 基因編輯, Cadmium,Rice,Nramp5,SNP,CRISPR, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 鎘 (Cd) 非植物的必須元素,卻容易經由必須元素通道累積在水稻穀粒中,鎘米會造成人體肝、腎的慢性毒害,因此控制穀粒的鎘含量對於糧食安全極為重要。在水稻吸收鎘的機制中,Natural resistance-associated macrophage protein 5 (OsNramp5) 運輸蛋白是水稻根部吸收鎘最主要的通道,過去的研究已知, OsNramp5 基因被離子束誘變法剔除時,可大幅降低水稻穀粒鎘含量,而利用基因編輯技術剔除OsNramp5 的水稻植株也同樣可獲得含鎘量極低的穀粒,這些結果顯示OsNramp5 是影響水稻穀粒鎘累積最關鍵的基因。本研究目的在使台灣本土水稻品種也具有低鎘累積特性,研究從兩個方向進行:(1)利用單核苷酸多型性資料庫篩選 OsNramp5 核苷酸具有變異的水稻品系,以作為育成低鎘水稻的種源;(2)應用基因編輯技術剔除台灣水稻品種的 OsNramp5 基因。從水稻單核苷酸多態性資料庫中,篩選出 50 個 OsNramp5 核苷酸具有變異的水稻品系,分析這些品系OsNramp5 胺基酸序列,發現有十個胺基酸序列改變的品系,具有穀粒低鎘累積潛力,其中 NSF-20、NSF-21 及 Nona Bokra 三個水稻都出現第 505 個胺基酸由丙胺酸 (Alanine, A, 基因編碼為 GCC) 變為蘇胺酸 (Threonine, T, 基因編碼為 ACC)之突變,分析鎘處理後 50 個品系幼苗地上部、根部的鎘累積量,發現 8 種鎘含量較低的品系。基因編輯試驗中,選擇台灣目前栽培面積最廣的台南 11 號、及研究資源最多的台農67 號進行基因編輯,針對 OsNramp5 基因第十個外顯子進行編輯,再利用基因定序篩選編輯成功的植株,未來可將進一步種植這些具有低鎘累積潛力的水稻於含鎘土壤,以評估具有低鎘累積潛力之水稻品系。 Cd is a toxic heavy metal and can lead to Cd-related diseases such as renal tubular dysfunction and bone disease. Cadmium (Cd) is not an essential element for plants. However, rice uptakes and accumulates Cd in grains by essential element’s transporters. The accumulation of Cd is a serious threat to human being since it can be concentrated in body through the food chain. Therefore, reducing the Cd content in grains is very important for food safety. Natural resistance-associated macrophage protein 5 (OsNramp5) is the main transporter which uptake Cd in roots. Knocking out of OsNramp5 has been reported to dramatically reduced Cd accumulation in rice grains. To generate low Cd accumulating Taiwanese cultivars, this research ainmed to carry out in two ways: 1. Screening for osnramp5 dysfunction mutants from single nucleotide polymorphism (SNP) database. 2. Genome-editing the OsNramp5 to Taiwanese cultivars. After screening SNP database from the rice 3000 genomes, 50accessions showed polymorphic genotypes on OsNramp5 genes. Analysis of amino acid sequences from 50 selected accessions showed that 10 accessions contained a change of amino acid. . In addition, three of ten accessions showed the same change at 505th amino acid, where Alanine (Ala, GCC) was substituted by Threonine (Thr, ACC). Examined of Cd concentrations on roots and shoots of rice seedlings showed that 8 lines have lower Cd content in roots or shoots. To generate genome-edited rice plants, two domestic rice cultivars, Tainan 11 (TN11) and Tainung 67 (TNG67), were used to precisely mutagenized the OsNramp5. Tainan 11 (TN11) is the most growing rice cultivars, while Tainung 67 (TNG67) has the most available research resources. Both cultivars’ 9th exon of OsNramp5 was chose for CRISPR/Cas9–based mutagenesis. Mutation was determined by DNA sequencing. Identification of OsNramp5 mutated accessions can be used as a parent for low Cadmium rice breeding. On the other hand, genome-edited rice plants can further screen low cadmium and transgene-free progenies. |
URI: | http://tdr.lib.ntu.edu.tw/handle/123456789/635 |
DOI: | 10.6342/NTU201903620 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 農業化學系 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-108-1.pdf | 1.37 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.