具有圓葉性狀的阿拉伯芥T-DNA嵌入突變株其功能性基因的研究

Abstract

植物葉片型態的發育(leaf ontogeny)是藉著複雜的機制來調控一連串基因表現的結果。因專一表現在葉片基因的產物大部分都是參與在光合作用或是氣體交換，因此欲分離及鑑定出可專一性調控葉片發育的基因並不容易。本研究利用T-DNA嵌入的方式建立阿拉伯芥突變株庫，再從其中篩選具有不正常葉型的突變株，來鑑定出調控葉片發育所需的基因。利用花序浸泡法進行基因轉殖，已建立了一個超過10,000株的突變株庫，並持續於T1及T2代中尋找具有葉型變異的突變株。本實驗即針對一個具有圓形葉性狀的突變株進行研究，探討其突變的基因與其所行使的功。此突變株暫時命名為RL1 (Round Leaf 1)。 RL1突變株除了葉型上的改變外，其植株高度及莢果長度，都和野生型植株有明顯的差異。其突變性狀和一些缺乏植物賀爾蒙brassionsteroid (BR)合成的突變株相似，但在處理BR後並不能使其回復成野生型性狀。對RL1突變株進行遺傳分析，其性狀的遺傳及抗除草劑BASTA能力的分離率均為3:1，表示RL1突變株是一個顯性突變。另外分別以兩種存在於T-DNA上的標誌基因BARR及pBluescript SK+的基因片段作為探針，進行南方氏雜合分析，推測 RL1突變株只有一個T-DNA嵌入，表示突變的性狀可能是由於單一基因改變所導致。利用質體回收法(plasmid rescue)得知T-DNA嵌入點是位於第一條染色體上Atlg 27120基因起始點ATG前195 bp的位置。此基因的功能推測可能是在參與細胞壁形成時所需要的半乳糖基轉移脢(galactosyltransferase)。針對RL1突變株進行北方雜合分析，顯示此基因的表現量有顯著的減少，因此推測RL1的突變性狀可能是此基因表現量的改變所導致。

The development of plant leaves is regulated by a very complicated system. In fact, the identification of genes that act as developmental controls in leaf ontogeny is problematic. Because two important processes that take place in the leaf, photosynthesis and the exchange of gases, both require the participation of a large number of gene products. Hence, a search for genes involved in the control of leaf morphogenesis by attempting to isolate gene products spatially restricted to this organ would identify those related to the execution of leaf functions rather than to the developmental mechanisms responsible for leaf. So, we choose the way to produce many Arabidopsis mutants with abnormal phenotype by T-DNA tagging. In our laboratory, we have produced more than 10,000 Arabidopsis T-DNA tagging mutants, and go on to search more mutants with abnormal phenotype in T1 and T2 generation. Now we focus on a special mutant, which has significance and genetic stable phenotype. Based on the abnormal round leaf phenotype, we named this mutant RL1, which means Round Leaf - l. Besides the leaf morphology, the height of plant, and the length of silique, are different from wild type plants. Then, we used Southern blotting techniques to prove that, the mutant was caused by only one T-DNA insertion and T-DNA flanking region was identified by plasmid rescue. Now, we found the mutant gene is Atlg 27120, which is predicted as a putative galactosyltransferase. It may play a key role involved in biosynthesis of cell wall. We suppose that, the abnormal leaf phenotype is caused by the downregulation of gene Atlg 27120.