輔仁大學
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狀態NC094FJU00105021
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學校名稱輔仁大學
系所名稱生命科學系
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學號493546235
研究生(中)李偉嘉
研究生(英)Wei-Chia Lee
論文名稱(中)微小水稻基因 tr1 的定位選殖
論文名稱(英)Positional cloning of TINY RICE Gene, tr1.
其他題名
指導教授(中)林彥蓉
指導教授(英)Yann-Rong Lin
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國圖全文開放日期.2007.01.01
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學位類別碩士
畢業學年度94
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語文別中文
關鍵字(中)定位選殖 水稻 微小
關鍵字(英)positional cloning rice dwarf
摘要(中)水稻株高為多基因遺傳性狀,由「綠色革命」成功培育半矮株小麥與水稻高產量品種的經驗得知矮株與產量的提昇具有相關性。時至今日,許多與水稻株高相關的基因被選殖出來,更有學者以遺傳工程方式結合提升產量與降低株高的基因培育高產量的新品種水稻。本實驗所使用之微小株水稻(Tiny Rice),為日本農業生物資源研究所Hirohiko Hirochika博士以組織培養方式刺激水稻內生性跳躍子Tos17插入剔除O. sativa ssp japonica cv. Nipponbare之眾多微小株之ㄧ,編號NC5171,由組織切片觀察到微小株之莖頂芽組織型態上較野生株尖銳、瘦長。tr1基因並未被Tos17標籤住,定位選殖法便成為選殖tr1基因的首選。以NC5171與Kasalath雜交所得的F2群體,經卡方檢定野生株與微小株之比例,可推知此一微小水稻性狀應為一隱性基因所調控。使用2,157棵隱性同型合子,以公開網路資源所取得之4個SSR及自行設計之7個Indel分子標幟將tr1基因定位至第八條染色體長臂尾端介於CH811至RM4997兩分子標幟之間,相距0.058 cM,為100.1 kb之標的區間,共有22個預測之候選基因。經由RT-PCR已將候選基因減少至8個,已完成定序的四個基因序列經BLAST比對Nipponbare序列後並無發現突變,暫時排除為tr1的可能性。將持續進行之工作:(1)組織切片之觀察,釐清 tr1基因影響之細胞及組織;(2)針對受影響之組織觀察tr1基因之表現情形;(3)繼續未完成之定序工作,找出突變發生之位置與型態。
摘要(英)Plant height is regulated by several genes and also influenced by environment. From the experience of “Green revolution”, which was successfully bred of the high- yield wheat and rice cultivars with semi-dwarf, we understand that the short structure is correlated to yield improvement. Up to date, more and more plant height genes have been cloned, and some researchers even incorporate high-yield genes and plant height genes by genetic engineering to establish high-yield varieties. The rice extreme dwarf mutant, Tiny Rice, is one of the Tos17 induced mutants with series number NC5171, which is created by Dr. Hirohiko Hirochika, National Institute of Agrobiological Sciences, Japan. The shape of shoot apical meristem (SAM) in the mutants is sharper than which in the wild type from the anatomy of the young seedling stage. The dwarf phenotype of NC5171 is affected by a single recessive gene named tr1, because the segregation ratio of wild type to mutants in the F2 families of NC5171 × Kasalath is obeyed the one single medelian gene by χ2 (goodness-of-fitt) analysis. Since tr1 is not tagged by the Tos17, the “positional cloning” approach has been employed to clone the tr1 gene. In total of 2,157 recessive mutants presumed as recessive homozygotes had been genotyped with four SSR markers and seven Indel markers for high-resolution mapping. The tr1 gene is mapped between marker CH811 and RM4997, which is estimated as 0.058 cM corresponding to 100.1 kb. There are twenty-two candidate genes in the target region after annotation. The candidate genes have been reduced to eight by the RT-PCR analysis of mRNA isolated SAM and surrounding tissues. Four candidate genes might be excluded due to no detected variation sites after multiple sequence alignment against wild type of Nipponbare genomic sequences. In the future, there will be three aims to do: (1) Histological observation: finding out the cells and tissues which is affected by tr1 in detail. (2) The tr1 expression: Understanding where and when the gene expression by RT-PCR analysis. (3) Accomplishment of the sequencing of the candidate genes: Identifying the mutation site and the mutation type. Thus, we can elucidate the tr1 function at molecular and biochemical levels.
論文目次目錄 i 表格目錄 iii 圖目錄 iv 中文摘要 v 英文摘要 vi 前言 1 水稻之栽培與重要性 1 水稻株高之研究 3 增進水稻產量之研究 7 水稻遺傳與基因體研究 8 水稻功能性基因體研究 10 定位選殖基因 15 研究目標 19 材料與方法 20 實驗材料 20 形態觀察 20 組織切片標本之形態觀察 21 基因型鑑定與連鎖分析 22 以序列分析觀察野生株與微小株tr1之基因序列差異 25 標的染色體區間之DNA定序 27 以RT-PCR篩選候選基因 29 結果 33 型態觀察 33 種子外型之觀察 33 幼苗型態之觀察 35 組織切片標本之觀察 39 精細定位tr1基因 43 tr1基因在F2子代之分離情形 43 連鎖分析 45 tr1之物理圖譜 48 候選基因之篩選 48 標的染色體之註解 48 以RT-PCR方法篩選候選基因 51 候選基因之定序 56 討論 57 型態觀察 57 精細定位 58 Indel分子標幟之搜尋條件與運用於F2子代基因型鑑定之情形 58 精細定位之解析度 59 以RT-PCR方式篩選候選基因 60 以定序方式鑑定候選基因 62 未來之實驗 63 參考文獻 64 附錄 84 附錄一、吉貝素之生合成途徑。 84 附錄二、油菜素內酯之生合成途徑。 85 附錄三、NC5171/Kasalath之F2種子數量預估以及分離率測試 86 附錄四、NC5171野生型與微小株種子外型之觀察。 87 附錄五、自行設計之Indel分子標幟 88 附錄六、以定序之染色體片斷與基因相關位置圖 89 附錄七、blastn比對之定序資料。 90 附錄八、候選基因功能概述 109
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