利用阿拉伯芥T-DNA基因功能活化種子庫篩選耐缺氮突變株

Screening of Arabidopsis Nitrogen Starvation Tolerance Mutant from T-DNA Activation Tagging Population

李其蓉(Chi-Rong Lee) ； 侯新龍(Shin-Lon Ho)

《嘉大農林學報》 7卷2期 (2010/08) Pp. 46-57

https://doi.org/10.29891/JAFNCU.201008.0004

摘要

本研究主要藉由阿拉伯芥T-DNA標的基因功能活化(T-DNA activation tagging)種子庫來篩選耐缺氮突變株，希望能獲得參與氮素訊息傳遞的調控基因。經缺氮(-N)培養基的篩選，共獲得2個可能的耐缺氮的基因活化突變株(Nitrogen starvation tolerance, nst)，分別為nst1及nst2。以bar基因為探針進行南方氏轉漬雜合分析證明這兩個突變株為相同的轉殖系，且染色體具有2個不同的T-DNA 插入位點。利用Plasmid rescue的方法，選殖出其中一個T-DNA的插入序列，經由DNA序列分析的結果，證明插入點位於1號染色體一未知功能的鋅手指(zinc finger)轉錄因子基因(AtZF1)，距離轉譯起始點上游1242核苷酸的位置。RT-PCR實驗證明突變株AtZF1基因的表現高於野生型(wild type, WT)，推測AtZF1可能受到35S加強子(4×35S enhancer)的活化表現，進而促進幼苗植株的耐缺氮功能。未來的研究將以過量表現的方法，將AtZF1基因轉殖到WT中，以確定AtZF1基因功能是否與阿拉伯芥幼苗的耐缺氮性有關。

關鍵字

阿拉伯芥 ； T-DNA基因功能活化標誌 ； 耐缺氮

並列摘要

The aim of this study is to screen genes involve in the nitrogen signaling pathway from an Arabidopsis T-DNA activation tagging mutant library under nitrogen free (-N) condition. Two putative mutants were selected, nst1 (nitrogen starvation tolerance 1), and nst2. Using Bar gene as a probe, Southern blot hybridization showed that two mutants are originally from the same transgenic event, and there are two T-DNA insertion sites on the chromosome. We have isolated one of the sequences adjacent to the right border from nst1 plant by plasmid rescue. DNA sequence analysis demonstrated that the T-DNA insertion position was located at chromosome 1, which was 1242 bp upstream from the translation start site of a putative zinc finger transcription factor gene (AtZF1). The gene expression of AtZF1 in nst1 was higher than that of WT according to the RT-PCR result, suggesting that AtZF1 gene expression was activated by 4× 35S enhancers, thus result in nitrogen starvation tolerance. Future work will be concentrated on the over-expression of the AtZF1 gene in WT plant to verify its possible function in regulating nitrogen signaling pathway.

並列關鍵字

Arabidopsis ； T-DNA activation tagging ； Nitrogen starvation tolerance