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Molecular cloning and functional analysis of GIGANTEA orthologues from various plant species
|關鍵字:||GIGANTEA;GIGANTEA;woody plants;flowering tome;oxidative stress;木本植物;開花時間;過氧化逆境||出版社:||生物科技學研究所||摘要:||
自木本植物洋紫荊（Bauhinia purpurea）老化葉片選殖所得之BpGI，乃是阿拉伯芥晚開花基因GI之同源基因。此一結果顯示AtGI/BpGI應不僅參與開花，亦與葉片老化相關。BpGI mRNA為常態表現，在洋紫荊發育過程與全年十二個月份中皆測得其表現，並在秋冬達到相對高峰。經由進一步分析得知，該基因為生理時鐘所調控，一天中表現高峰為光照後8~12小時，而在清晨時最低。當異位表現BpGI或AtGI含四個NLS之C端於晚開花突變種gi-1時，GI之下游基因CONSTANS（CO）明顯地被調控，且gi-1突變種之晚開花現象被彌補，因而開花正常。有趣的是，經由yeast two hybrid analysis得一與BpGI蛋白質結合之葉綠素a,b結合蛋白質四號前驅物（CAB 4 precursor），其突變種亦呈現晚開花之現象。有鑑於曾有研究指出gi突變種會抗巴拉刈（paraquat）所引起之過氧化逆境，故植物長壽與抗逆境之關聯應是密不可分。經由偵測阿拉伯芥中與抗過氧化逆境的兩個重要基因CAT2與CAT3得知，在gi-1突變株中此二基因之表現量遠高於在野生種中之表現，其中CAT3之表現量甚至高至無法分辨其原有之日夜節律。另外，在葉片分離之老化測試試驗中，gi突變種之葉片在離體十五天後仍未黃化。進一步之研究更發現gi較能抵抗青枯病菌感染，更甚至能抵擋冷逆境處理等。再者，將生長於短日照30天及長日照7天之gi與野生株之cDNA，作差異性篩選與微陣列分析，結果亦發現gi突變株中許多與冷誘導與抗逆境相關基因有持續高表現之現象。綜上，GI在開花與葉片老化等抗過氧化逆境的調控上應扮演一全新角色。
BpGI, isolated from senescent leaves of woody plant Bauhinia purpurea, is a homologue of Arabidopsis late-flowering gene GIGANTEA (GI). This revealed that GI/BpGI was involved in not only flowering but also leaf senescence. BpGI mRNA is expressed throughout development and can be detected all year round with relatively high expression during fall and winter. Further analysis indicated that the expression of BpGI is modulated by the circadian clock with the highest level at the 8 to 12 h after dawn and lowest at dawn. Ectopically expressed C-terminal part of either BpGI or AtGI that containing four putative nuclear localization signals (NLSs) significantly promotes flowering by up-regulation of the CONSTANS (CO), a downstream gene of GI, in late-flowering mutant gi-1. Interestingly, mutation in a gene, encoding for chlorophyll a/b binding protein 4 precursor homolog which showed strong interaction with BpGI protein in yeast two hybrid analysis , also caused a late-flowering phenotype in Arabidopsis. Since mutation in GI caused high tolerance to paraquat-induced oxidative stress, further exploring the link between late flowering and longevity in plants is necessary. The result indicated that a significant up -regulation of two catalase genes, CAT2 and CAT3, was observed in gi mutants. Furthermore, the circadian clock regulation of CAT3 was completely abolished in gi mutants. In addition, leaves removed from gi mutants did not show senescence up to 15 days after detaching. Mutation in GI gene also enhanced tolerance to cold stress and resistance to bacterial pathogen Ralstonia solanacearum. Interestingly, several cold and other stress induced genes were identified by screening the cDNA subtraction library for wild-type and gi mutants grown for 30 days under short day and then shifted to long day for 7 days . Our results discovered the novel roles for GI in regulating leaf senescence and flowering in response to oxidative stress.
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