https://scholars.lib.ntu.edu.tw/handle/123456789/139492
標題: | 利用阿拉伯芥 T-DNA Knockout 突變株 , 研究調控葉部形態發育之功能性基因(2/3) | 作者: | 靳宗洛 | 關鍵字: | 葉部形態;T-DNA 突變株;基因釣取;creasd leaf1-1D;creasd leaf1-2D | 公開日期: | 31-七月-2004 | 出版社: | 臺北市:國立臺灣大學植物科學研究所 | 摘要: | 藉由pPZP202:BAR:SK 此全新、高便利性及高效率的T-DNA tagging 載體, 我們篩選出許多葉部形態在T1 代就與野生型有差異的阿拉伯芥突變株。在此, 我們針對二株性狀類似的葉型突變株進行研究,分別命名為creased leaf 1-1Dominant(crl1-1D)及creased leaf 1-2D(crl1-2D),它們的葉片具有皺縮、 由外向內反捲且較為細長等突變性狀。此外,它們的生育力也較低,同時在它們 自交子代中會出現只長出第一對真葉後即停止其他真葉發育的變異株。由 Southern blot 分析得知這二個突變株各有二個T-DNA 嵌入,而嵌入位置並不完 全相同,顯示這兩個突變株是各自獨立的,利用Inverse PCR(IPCR)找出其中 一個T-DNA 嵌入位置,發現二個突變株各有一個T-DNA 嵌入At2g36310 基因的 下游,而且僅相差307 bp,此基因暫先命名為CRL1。經序列分析推測CRL1 可 能是一個次黃苷/尿嘧啶核苷水解酶,由Northern blot 分析顯示在crl1-1D 和 crl1-2D 突變株中CRL1 基因的表現量均高於野生型。因此,在野生型中大量表 現CRL1 基因,結果在部份35S::CRL1 轉殖株中發現有長出第一對真葉後即停止 其他真葉發育的轉殖株,這與阿拉伯芥wuschel (wus) 突變株類似,但是並沒有 重現葉片皺縮的性狀。而葉部發育被抑制的轉殖株中WUS 基因並沒有表現。因 此我們推測crl1-1D 和crl1-2D 變異株的葉片皺縮型態也許和CRL1 並沒有直接 關聯,但它的表現量可能對植物頂端分生組織的活性有影響,使葉片發育異常, 但和葉部型態可能沒有直接的關聯。 By means of a de novo T-DNA tagging vector pPZP202:BAR:SK with high convenience and efficiency, we have screened many Arabidopsis T1 T-DNA knockout lines, whose leaf morphology differs obviously from that in wild-type plant. In this report, we have identified two gain-of-function mutants, which were selected in the T1 generation, crl1-1D (creased leaf 1-1 Dominant) and crl1-2D with a wrinkled curling inward and narrow leaf phenotype. In addition, the fertility was lower than wild type and some of the self-crossed progenies ceased to develop further after the first two leaves were initiated. Using Southern blotting analysis, two T-DNA insertions with different Southern patterns were detected in each line, indicating that crl1-1D and crl1-2D were independent T-DNA tagging lines. Inverse PCR (IPCR) identified that one of the T-DNA insertion sites in each mutant. Noteworthily, the T-DNA insertion site both are located at the down-stream region of At2g36310 (CRL1), and is distanced from each other by 307 bp, which encodes a putative inosine-uridine nucleoside hydrolase (IUNH). Northern blotting analysis using CRL1 coding region as a probe shown that the transcript levels were higher in mutants than that in wild type. To recapitulate phenotype of the crl1-1D and crl1-2D in wild type, transgenic plants with CRL1 overexpression were generated. However, no transgenic plant with wrinkled leaf was observed, instead some 35S::CRL1 seedlings showed phenotype similar to the loss-of-function mutant wuschel (wus). The WUS transcripts were not detected by RT-PCR analysis in 35S::CRL1 lines with the wus phenotype, instead a shoot apical meristem (SAM) growth suppression was found. From these results, we suggest that overexpression of the CRL1 might affect the SAM activity rather than leaf morphology. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/17461 | 其他識別: | 922311B002025 | Rights: | 國立臺灣大學植物科學研究所 |
顯示於: | 植物科學研究所 |
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