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標題: 擬南芥中兩個CONSTANS—like基因的分子選殖與功能分析
Characterization and Functional Analysis of Two CONSTANS-like Genes from Arabidopsis thaliana
作者: 蔡人杰
Tsai, Ren-Jie
關鍵字: 擬南芥
出版社: 農業生物科技學研究所
摘要: 擬南芥為一年生草本植物,在進入生殖生長前會經過幾個不同的營養生長時期。而目前對於調控擬南芥由營養生長進入生殖生長(開花時間)的決定因子研究方面,已有許多參與抑制或促進開花的早晚開花基因被選殖出來。其中晚開花基因CONSTANS(CO)是一具有zinc finger區域的轉錄因子蛋白,被認為與促進開花有關。本實驗依據序列的相似性,從擬南芥中選殖出兩個CO -like的基因,分別命名為ACOZ1和ACOZ2(Arabidopsis CO -like Zinc finger gene 1 and 2)。ACOZ1位於一號染色體上,有三個表現子(exon),可轉譯出一含299個氨基酸的蛋白。而ACOZ2則位於二號染色體上,同樣有三個表現子,可轉譯出一含237個氨基酸的蛋白。ACOZ1及ACOZ2的mRNA在播種後皆有表現增加的情形,且在營養葉、腋生葉、及花上都可偵測到其存在。另外ACOZ1及ACOZ2的表現在早開花突變elf1, 2, 3中皆有上升的現象,此表現情形和CO非常類似。進一步的研究發現ACOZ1及ACOZ2的表現也會受冷、熱及高鹽逆境的刺激而上升。為了探討這兩個基因在功能上是否如同序列及表現情形一樣和CO相類似,以CaMV 35S啟動子驅動的ACOZ1正反向ORF及正向染色體片段之構築體,被轉入野生種及晚開花突變種之擬南芥中。分析後發現轉殖ACOZ1 ORF正反向構築體之野生種,在開花時間上都沒有明顯的變異,而轉殖ACOZ1 ORF正向構築體的晚開花突變種gi,其植株的開花時間有提早的現象。推論ACOZ1基因之功能應與CO相似,但其作用能力應較CO為弱,也就是在開花過程中其作用為扮演輔助CO之角色。另外亦發現轉殖ACOZ1 ORF正向構築體的植株其抗鹽性明顯增加,而轉殖ACOZ1 ORF反向構築體的植株則出現短根的性狀。帶有ACOZ2正反向ORF及正向染色體片段之構築體亦構築完成,以待將來進行轉基因植物功能性之分析。
The control of this vegetative-to-reproductive transition (or flowering time) has been extensively studied in Arabidopsis. Many different early- and late-flowering genes involved in either promoting or delaying the timing of the rosette-to-inflorescence transition have been cloned and characterized. Late-flowering gene CONSTANS (CO), encoded a zinc finger protein, has been thought to act as 1a transcriptional factor to promote flowering. Based on sequence similarity, two genes, ACOZ1, and ACOZ2 (Arabidopsis CO-like Zinc finger gene 1 and 2), showed homology to CO in Arabidopsis genome were cloned and characterized in this study. ACOZ1 gene, contained three exons on chromosome 1, encodes a putative 299 amino acid protein. ACOZ2 gene, contained three exons on chromosome 2, encodes a putative 237 amino acid protein. ACOZ1 and ACOZ2 mRNA were detected early and were increased gradually after germination. ACOZ1 and ACOZ2 mRNA can be detected in rosette leaves, cauline leaves and flowers after flowering. The expression of ACOZ1 and ACOZ2 mRNA was upregulated in early-flowering mutants elf1, 2, 3 and was induced by cold, heat and salt treatments. The expression pattern for ACOZ1 and ACOZ2 was very similar to that observed for CO. To further explore if the sequence and expression likeness is coupled to the functional similarity between ACOZ1, ACOZ2 and CO, functional analysis of ACOZ1 and ACOZ2 through transgenic plants is performed. Sense or antisense cDNA or genomic DNA for ACOZ1 were ectopically expressed in wild-type and late-flowering mutant Arabidopsis plants. Further phenotypic analysis indicated that transgenic wild-type plants were phenotypically indistinguishable from untransformed wild-type plants whereas the flowering time was reduced in transgenic late-flowering gi mutant plants. Our result indicated that ACOZ1 gene was function similar to CO in regulating flower transition although in a relative weak manner. In addition to regulate flowering time, ectopic expression of ACOZ1 significantly increased the salt tolerance in transgenic plants. By contrast, ectopic expression of antisense of ACOZ1 significantly reduced the length of the roots in transgenic plants. Constructs contained sense and antisense ACOZ2 were also constructed for further functional analysis through transgenic approach..
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