具有捲曲葉型和延遲開花性狀的T-DNA 插入突變株之功能性基因研究

Abstract

葉子是植物外表最顯著的器官，並且在光合作用、呼吸作用和氣 體交換扮演重要角色。然而到目前為止，關於葉部發育的細節並不十分清楚。因此，本實驗室使用正向遺傳學來分析在阿拉伯芥的T-DNA突變株庫中，具有葉部發育缺失突變株並找出影響葉型的基因。本實驗室利用T-DNA 嵌入載體 pPZP202:BAR:SK 來產生突變株並進行篩選，在本研究中我們從T2 的突變株庫找到一株具有捲曲葉型和延遲開花性狀的突變株，並命名為R1-2。將突變株和野生型作側交，發現只有在F2 子代才發現具有突變性狀的植株，因此推測R1-2 為一株隱性突變的植株。經由南方墨點法和IPCR 分析，在R1-2 突變株中有二個T-DNA，並以SK 相連的方式嵌在At3g22970 上游約2.5kb 處。經由RT-PCR 和北方墨點法比較野生型和R1-2 間At3g22970的表現得知：在R1-2 中At3g22970 表現量下降，因此推測R1-2 的性狀可能和At3g22970 表現量下降有關。At3g22970 是一個位在葉綠體並具有DUF506 區域的表現蛋白，在阿拉伯芥中共有十四個基因具有此種區域，到目前為止依然不知其功能。使用RT-PCR 和北方墨點法可發現At3g22970 的表現在一天中具有韻律性，而且在全株植物的器官均可表現，特別在花部表現最多。然而對於At3g22970 的功能依然不了解，因此我們觀察over-expression 和 knock-out 的突變株來了解At3g22970 在植物的生長發育中扮演何種功能。而在這些over-expression 轉殖株中, 有些轉殖株的性狀和野生型相同，有些轉殖株則具有開花延遲的性狀並降低At3g22970 的表現量， 我們推測具有開花延遲性狀的over-expression 轉殖株可能是由於大量表現At3g22970 進而共同抑制其表現，因此產生延遲開花的性狀。在另一方面，knock-out 的突變株也有一株具有延遲開花的性狀。由以上結果可知，At3g22970可能扮演某種角色在開花時間的控制上。

Leaves are the most noticeable plant organs and play important roles in photosynthesis, respiration and gas exchange. However, the details of leaf development remain unclear. Therefore, our laboratory used forward genetics to identify the mutants with leaf development defect from T-DNA tagging pools in Arabidopsis. We use a T-DNA tagging vector, pPZP202: BAR: SK, to generate mutants and to screen for morphological mutants. In this study, we have identified a mutant, R1-2, which were selected in the T2 generation, with curled leaves and delayed flowering phenotypes. After R1-2 was crossed to the WT, none of the R1-2 phenotypes was observed in the F1 but were found in the F2 population. Therefore, it was suggested that R1-2 was a recessive mutant. Southern blotting analysis and inversed PCR identified two T-DNA linked together and inserted in the 2.5kb upstream of the At3g22970. Using RT-PCR and Northern blotting assay, we showed that the transcripts of At3g22970 were down-regulated in mutant, implicating that the phenotypes of R1-2 may be resulted from down-regulation of At3g22970. At3g22970 was predicted as a protein containing DUF506 domain and located in the chloroplast. There are 14 genes that contain DUF506 domain in Arabidopsis thaliana and all with unknown function. Using RT-PCR and Northern blotting assay, it was detected that the expression of At3g22970 appeared rhythmic, and expressed in the whole plant, especially in flowers. However, the function of At3g22970 is still unknown. We observe the phenotype of the transgenic plants by over-expressing or knock-outing the At3g22970 to address its function in Arabidopsis. Among these over-expression lines, some lines with delayed flowering phenotype had reduced expression of At3g22970, but others were the same as WT. The correlation between delayed flowering time phenotype and reduced expression of At3g22970, indicating that the phenotype was caused by co-suppression of the over-expressed At3g22970. Additionally, one of the SALK knock-out lines, SALK-085910, also exhibited a delayed flowering phenotype. Therefore, At3g22970 might play a role to control flowering time.