水稻（Oryza sativa L.）是世界上重要的糧食作物之一，利用T-DNA插入性突變族群進行水稻功能性基因研究目前已有很好的成果。而T-DNA轉殖水稻時需經癒傷組織，容易產生體細胞變異，故突變體性狀和T-DNA插入基因的相關性不高，大約5-10％，而造成研究上的困擾。本研究應用TILLING（targeting induced local lesion in genomes）了解台農67號和Nipponbare在蠟質基因（6297 bp）上的SNP（single nucleotide polymorphism） 。在台灣Hsing等（2007）利用T-DNA轉殖水稻台農67號， 產生了TRIM（Taiwan rice insertion mutants）族群，因此本研究應用TILLING快速偵測點突變特性，以蠟質基因（Waxy）為模式，篩選經T-DNA突變的TRIM族群，探討體細胞變異率及變異的型式。
實驗結果指出台農67號和Nipponbare品種的在蠟質基因上並沒有多型性。在200個TRIM水稻品系，共3000個單株中，篩選蠟質基因上的體細胞變異。有五個突變品系在蠟質基因上有多型性，其中有一個突變品系有兩個多型性，因此共有六個多型性，且都是屬於核苷酸置換 （substitution） 。六個SNPs位在啓動子和5，-UTR。本研究在3000單株×蠟質基因6297 bp＝18,891,000 bp中，找到了六個SNPs，變異率為3.1×10-7。

Rice（Oryza sativa L.）is one of the most important crops in the world. T-DNA has been used successfully in rice functional genomics studies. For rice transformations, the integration of T-DNA occurred at the callus tissue, as a consequence, somaclonal variations might be generated during cultured period. The tagging efficiency of T-DNA induced population is relatively low, that is, about 5-10％ of the phenotype co-segregate with the T-DNA integration site. In the present research, TILLING, targeting induced local lesion in genomes, a novel technique with high throughput discovery of single nucleotide changes, was used to detect SNPs, single nucleotide polymorphism, in Waxy locus between two japonica varities, Tainung 67 and Nipponbare. In Taiwan, Hsing et al.,（2007）used T-DNA transform to build up a mutant population using an elite local variety Tainung 67 and designated TRIM, Taiwan Rice Insertion Mutants. In the present research, TILLING was used to reveal Waxy locus muataions in TRIM population, in order to characterize the frequency and type of somacloal variations.
No SNPs are present in Waxy locus（6297 bp） between Tainung 67 and Nipponbare cultivars. For 200 TRIM lines, or 3000 individual plants, five lines consisted somaclonal variations in Waxy locus. One of these lines contained two SNPs, thus, totally 6 SNPs were detected. All SNPs were belong to nucleotide substitution, and they located in the promoter or 5，-UTR region in Waxy gene. To conclude, 6 nucleotide substitutions was found , after screening 3000 plants × 6297 bp Waxy gene. The mutation frequency is estimated as 3.1×10-7.

目錄 i
圖次 iii
表次 iv
中文摘要 v
Abstract vi
前言 1
前人研究 3
一、 TRIM T-DNA突變水稻族群介紹 3
二、 體細胞變異 5
三、 TILLING在植物上的應用 8
四、 水稻蠟質基因 11
材料方法 13
一、 試驗材料 13
二、 試驗方法 13
(一) 純化CEL I 13
(二) 萃取DNA 14
(三) 蠟質基因引子 15
(四) TILLING 15
(五) 定序 16
試驗結果 18
一、 CEL I 活性 18
二、 台農67號和Nipponbare的SNP 21
三、 TILLING在TRIM 水稻族群的應用 23
討論 31
一、 TILLING 在TRIM 水稻族群的應用 31
二、 TRIM族群體細胞變異探討 35
附錄一 38
參考文獻 44

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