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標題: 阿拉伯芥中AtNAC-like 14基因之功能性分析及sa7突變株之定性分析
Functional analysis of AtNACL14 gene and characterization of sa7 mutant in Arabidopsis thaliana
作者: 陳宏翊
Chen, Hong-Ie
關鍵字: NAC
出版社: 生物科技學研究所
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摘要: NAC (for NAM, ATAF1, 2, and CUC2)基因為一群植物特有的基因群。大部分的NAC蛋白在其N端均含一個約為150胺基酸的高保守性DNA-binding domain以及NLS (localization signal sequence)序列。以阿拉伯芥為例,NAC蛋白參與許多植物生長和發育的過程,例如cell cycle的控制、生長荷爾蒙訊息的傳遞、花器的發育和頂端分生組織的形成等。為了探討NAC基因的功能,我們將阿拉伯芥NAC2 subgroup之AtNACL14啟動子(promoter)後面加上一個GUS報導基因轉殖入阿拉伯芥,進行GUS組織活性分析。結果顯示GUS高表現在根、子葉、芽頂端分生組織及葉。此外,目前在阿拉伯芥中,有一群NAC蛋白C端被預測出具有α螺旋的穿膜功能域,此功能域被認為與蛋白結合在膜上的能力有關。分析大量表現全長AtNACL14基因(35S::AtNACL14)與刪除α螺旋的穿膜功能域(35S:: AtNACL14ΔTM),以及去除C端區域的35S:: AtNACL14ΔC轉基因植物分析結果中發現,大量表現AtNACL14與AtNACL14ΔC之轉基因植物外表型與野生型阿拉伯芥無異。令人驚訝的是,大量表現AtNACL14ΔTM之轉基因植物外表型顯示出植株矮化和葉的扭曲。综合上述結果顯示,具有穿膜功能的NAC轉錄因子對於植物的生理功能具有舉足輕重的影響。本研究另外分析一個具有穩定性狀的T-DNA突變株,sa7突變株它的性狀是營養葉呈現圓形,而且葉面相對於野生型阿拉伯芥較光滑。藉由IPCR的鑑定發現,此T-DNA是位在距離At5g24590上游1572個bp的位置。分析sa7突變株的基因型,發現其是以同型接合子(homozygote)的基因型存在,表現量分析,發現At5g24590的表現量在突變株中較野生型阿拉伯芥低,推測可能是At5g24590的表現受到抑制造成sa7突變株之性狀,因此針對此基因進行功能性分析,分析結果尚未篩選到大量表現At5g24590轉基因植物,而在大量表現At5g24590 RNAi的轉基因植物,其性狀與野生型阿拉伯芥無異,因此這可能暗示著我們T-DNA插入的位置,可能造成包含At5g24590以及其它基因之表現也受影響,因此才共同造成sa7突變株的性狀。
NAC genes (for NAM, ATAF1, 2, and CUC2) are plant specific gene family. Most NAC proteins contain one highly conserved N-terminal DNA-binding domain, consisting of approximately 150 residues and a nuclear localization signal sequence. Some Arabidopsis NAC members involve a variety of plant growth and development processes, such as cell cycle control, growth hormone signaling, floral development, and apical meristem formation. To investigate the function of gene, the promoter of AtNACL14 in NAC2 subgroup was fused with GUS reporter gene, transformed into Arabidopsis and GUS activity analyzed. The results indicated that AtNACL14 were highly expressed in roots, cotyledon, shoot apical meristem and leaves. In addition, NAC family in Arabidopsis containing strong α-helical transmembrane motif (TM) in their C-terminal regions and are predicted to be membrane-associated. Transgenic plants ectopic expression of either full-length of AtNACL14 (35S::AtNACL14) or the truncated AtNACL14 construct (35S:: AtNACL14ΔC) were analyzed. The results indicated that 35S::AtNACL14 and 35S:: AtNACL14ΔC no show any detectable phenotypic changes. Surprisingly, the 35S::AtNACL14ΔTM transgenic plants exhibited severe phenotypic alterations such as dwarfism and curled leaves. This result revealed that membrane release is essential for the function of NAC MTFs (membrane-associated transcription factors). An Arabidopsis T-DNA insertional mutant caused circle and smooth rosette leaves was characterized in this research. Through inverse PCR (IPCR), it has been found that the T-DNA was inserted in 1572 bp of 5' UTR from start codon of At5g24590. All the sa7 mutants are homozygotes for T-DNA insertion and At5g24590 mRNA were down-regulated in the sa7 mutants. This result revealed that the phenotype of sa7 mutant may be caused by the repression of At5g24590 after T-DNA insertion. To explore this possibility, sense and RNAi of At5g24590 cDNA driven by 35S promoter were transformed into Arabidopsis and phenotypic analyzed. Notably, we could not obtained 35S::At5g24590 transgenic plants by selected solid MS medium. The indicated that RNAi plants were phenotypically indistinguishable from wild-type plants. It implied that T-DNA insertion resulted in contain At5g24590 and other gene were affected.
其他識別: U0005-1108200823414100
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