中文摘要 為了了解植物耐受高溫的遺傳控制因子及其生理機制，我們用化學突 變劑( EMS )誘導阿拉伯芥點突變產生，並進而篩選出一株對熱缺乏耐受 性之突變種，稱之為hit1( heat intolerance ) 。這是一種典型從具遺傳性之 生理性狀找出相對應之基因及其分子作用的研究方式，亦即所謂的向前式 遺傳學分析法( forward genetics )，為功能性遺傳學研究法中的一種。因 hit1 之性狀為點突變所造成的，故其功能性基因須以遺傳圖譜為基礎來進 行定位選殖( map-based cloning, MBC ) 。其做法是先將Columbia 生態 型之hit1 突變種與Landsberg 生態型之野生種雜交，之後的F1 自交得F2 世代，F2 世代中帶有hit1 性狀之個體為hit1 定位時所需之分離族群 ( segregation population )。以hit1 之突變性狀，37℃處理四天後會死亡的 原則篩選， 再經簡單序列長度多型性( simple sequence length polymorphism , SSLP )及限制酵素切割擴增片段多型性( cleaved amplified polymorphic sequence , CAPS )兩種對生態型具專一性之分子標記進行定 位。本研究共設計了26 組SSLP 分子標記，7 組CAPS 分子標記。在檢 測過3987 株F2 世代的hit1 突變種之後，推得hit1 基因坐落在第一條染色 體， AGI map 18767.3 kb ~ 18810.2 kb 之42.9kb 範圍內，達到MBC 所需 之定位目標。 Abstract In order to understand the mechanism of plant crop heat stress, we screened a mutant that is difficult to tolerant heat stress called “hit1” (heat intolerance) inducible by chemical mutagenesis, EMS, causing a point mutation. Forward genetic is one of functional genetics approaches, and it is underlying a mutant with a desirable genetic phenotype to an identified mutation in a gene and to analysis specific gene’s molecular biology function. We use forward genetic to approach “hit1” that was a point mutation, so we want a cloning of sequencing mutant-define gene by map-based coloning (MBC). A hit1 mutant with Columbia ecotype (P1) and a wild type with Landsberg erect ecotype (P2) were parent to get recombinant inbred lines (F2). It has hit1 mutant specific phenotype lines of F2 generation that is a segregation population to provide hit1 genetic mapping. The definition of hit1 mutant phenotype was death at 37℃ in 4 days to screen mutants from F2 generation as a segregation population then make hit1 gene genetic mapping, and we designed 26 pairs primers of simple sequence length polymorphism (SSLP) markers and 7 pairs primers of cleavage amplify polymorphisms (CAPS) markers for genetic analysis. We suggest that hit1 gene locus in AGI 18767.3kb ~ 18810.2kb, total 42.9kb regions, and approach the fine-scale mapping of MBC.
