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|標題:||探討阿拉伯芥中E3 RING finger基因與文心蘭中CONSTANS (CO)-like基因調控植物發育及開花之特性
Characterization of an Arabidopsis E3 RING finger gene and an Orichid (Oncidium Gower Ramsey) CONSTANS (CO)-like gene in regulating plant development and flowering
|關鍵字:||Arabidopsis;阿拉伯芥;Oncidium Gower Ramsey;E. grandiflorum;ubiquitin;flowering gene;CONSTANS-like;RING5;文心蘭;洋桔梗;泛素;開花基因;CONSTANS-like基因;RING5基因||出版社:||生物科技學研究所||引用:||參考文獻 施靜芳(2004) Functional analysis and the application of NAC-like genes and GIGANTEA (GI) orthologues in regulating meristematic activity, flowering and senescence in plants. 國立中興大學生物科技研究所碩士論文。 廖珮君(2004) Characterization and functional analysis of genes regulating cell division and differentiation in Arabidopsis. 國立中興大學生物科技研究所碩士論文。 欒乃勳(2006) Molecular cloning and functional analysis of GIGANTEA (GI) orthologues from fern Adiantum capillus-venen and NAC-like genes from Arabidopsis. 國立中興大學生物科技研究所碩士論文。 Aida, M., Ishida, T., Fukaki, H., Fujisawa, H., and Tasaka, M. (1997). Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9, 841-857. Azevedo, C., Santos-Rosa, M.J., and Shirasu, K. (2001). The U-box protein family in plants. Trends in plant science 6, 354-358. 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植物由胚胎發育、營養生長及生殖生長，是受到各種不同的發育途徑與基因所影響。具RING finger domain的E3s接合酶(E3s ligase)，影響植物多方面的生理反應。本研究由阿拉伯芥中選殖出AtRING5基因進行功能性分析。藉由啟動子活性分析，進一步構築AtRING5基因的啟動子藉由驅動GUS報導基因的表現來呈現它們的表現位置。結果發現GUS明顯表現在分生組織附近的區域，且在新生組織中表現明顯，並隨著組織發育成熟而逐漸減少。經由生長素IAA誘導發根，發現GUS主要表現在新生側根分生組織，確定AtRING5與分生組織的發育有關。而在異位大量表現基因(sense)及抑制基因表現(antisense及RNAi)後，發現主花序發育受到影響、胚胎期子葉發育異常、根形態改變及根毛發育異常之現象，推測AtRING5具有調節早期生長發育的功能。植物由營養生長到生殖生長的過程中，受到許多途徑的調節，而光週期途徑中的CONSTANS (CO)基因扮演關鍵的角色。CO會誘導下游FT表現而促使開花。本研究由文心蘭及洋桔梗中選殖CO-like同源基因，文心蘭中選殖到的OnCOL1轉譯出328個胺基酸，並會受到黑暗而促進表現。異位大量表現OnCOL1於阿拉伯芥中，會有提早開花的現象。另外在洋桔梗中選殖到EgCOL1、EgCOL2及EgCOL3三個CO-like同源基因，EgCOL1由369個胺基酸構成，EgCOL3由320個胺基酸構成，而EgCOL2只有571 bp的片段。EgCOL1及EgCOL3在萼片、花梗及葉片皆有明顯表現。而此三個EgCO-like的功能性，需經由進一步基因轉殖至阿拉伯芥及洋桔梗中才能確認。
Plant development is regulated by many genes and pathways from embryotic phase, vegetative phase to reproductive phase. RING genes that contained a RING finger motif as E3 function have been thought to be involved in ubiquitination pathway in regulating plant development. AtRING5 with conserved RING domain encoded a 493 amino acid protein was identified in Arabidopsis. To further investigate AtRING5 function, promoter assay and transgenic approaches were used. Promoter assay by transforming constructs fusing the promoter of AtRING5 with report GUS gene in Arabidopsis indicated that AtRING5 was expressed in young shoot and root meristem and its expression was decreased during late stage of organ development. Furthermore, GUS was detected in the meristem of the newly formed lateral roots in Arabidopsis after IAA induction. These results indicated that AtRING5 may be a regulator in controlling the meristem formation. Transgenic plant that ectopic expressed sense, antisense and RNAi (RNA interference) of AtRING5 resulted in the alteration of inflorescence differentiation, cotyledon formation, root morphology and root hair development. This data supported that AtRING5 is functioning in regulation of meristem formation during early development stage. Many pathways regulated the flowering transition including circadian clock associated genes such as GI and CO (CONSTANTS) which play a mediated role in photoperiodic pathway of Arabidopsis. CO has been thought to activate its target gene FT (Flowering locus T) to promote the flowering in LDs. In this study, CO-like genes were cloned and characterized from Oncidium Gower Ramsey and Eustoma grandiflorum. The expression of Oncidium OnCOL1 that encoded a 328 amino acids protein was induced by dark treatment. In 35S::OnCOL1 transgenic Arabidopsis plants, acceleration in flowering time was observed. In addition, three CO-like genes, EgCOL1 and 3 encode proteins of 369 and 320 amino acid residues and EgCOL2 with 517 bp partial DNA sequence were cloned from E. grandiflorum. Constructs contained EgCOL genes were constructed for further functional analysis through transgenic approach in both Arabidopsis and E. grandiflorum.
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