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標題: 擬南芥中一EST clone與晚開花基因FLD關係的探討
The Investigation of the Relationship Between an EST Clone and Late-flowering Gene FLD from Arabidopsis thaliana
作者: 楊緒元
Yang, Shu-Yuan
關鍵字: Arabidopsis thaliana
development switch
flowering time
late-flowering gene
出版社: 農業生物科技學研究所
摘要: 在擬南芥 (又名阿拉伯芥,Arabidopsis thaliana) 中FLD基因的突變會造成極端晚開花的表現型。FLD基因被定位在阿拉伯芥的第三條染色體上臂12 cM處。一個1.4 kb長之EST (Expressed Sequence Tags) clone 34B2T7和FLD基因在染色體上的位置非常聯鎖。由南方墨點法顯示34B2T7在阿拉伯芥中為一單套基因。而由北方墨點法顯示34B2T7的表現是受到植物不同發育時期所調控。mRNA能在所有組織中被偵測到,包括營養葉、莖、花。在花序中的表現顯著的比其他組織高出許多 (約5倍以上)。而抽花序前營養葉的mRNA量也高於抽花序後之營養葉的mRNA量。這結果顯示34B2T7的表現模式是與理論上FLD基因的表現情況非常相似。另外,34B2T7的表現量在經水楊酸 (SA) 處理後12小時會被誘導出來,而經ABA及wounding處理後沒有影響。為再進一步研究34B2T7和FLD基因間的相關性,經由野生型Columbia植株的in planta vacuum transformation,我們得到包含34B2T7 sense和antisense構築體的轉基因植物。含有sense構築體的轉基因植物表現出較早開花的表現型,而antisense構築體的轉基因植物則表現出延遲開花的表現型。這結果強烈的暗示這34B2T7可能由FLD基因所表現之cDNA產物。更進一步,由分析genomic DNA序列顯示,這encoded 34B2T7的基因包含4個exons和3個intron。由此34B2T7序列推測出一含335胺基酸之蛋白質。經比對顯示和幾種其他植物 (水稻、馬鈴薯、小麥) 的basic chitinase有40%一致及60%的相似。這表現34B2T7產物的構築體已經構築成功,以便未來進行其蛋白質的功能分析。
Mutation of FLD gene in Arabidopsis caused an extremely late-flowering phenotype. FLD gene has been mapped on the long arm of chromosome 3 in about 12 cM of Arabidopsis. An EST (Expressed Sequence Tags) clone 34B2T7 closely linked to FLD gene was characterized here. Genomic Southern hybridization indicates that 34B2T7 is encoded by a single copy gene in Arabidopsis. RNA blot analysis indicates that the expression of 34B2T7 is developmentally regulated. The mRNA was detectable in all organs tested, included rosette leaves, shoots, and flowers. However, a significantly higher mRNA accumulation (about 5-fold more) was observed in shoots than in other organs examined. The mRNA level is also higher in leaves before bolting than in leaves after bolting. This result indicates that the expression pattern for 34B2T7 is very similar to the hypothetical function of FLD gene. In addition, the expression of 34B2T7 is induced by salicylic acid (SA) 12 hr after treatment whereas abscisic acid (ABA) and wounding have no effect on the induction of 34B2T7. To further investigate the relationship between 34B2T7 and FLD gene, transgenic plants containing either sense or antisense constructs of 34B2T7 were generated through in planta vacuum transformation of wild-type Columbia plants. Transgenic plants with sense construct exhibited early- flowering phenotype whereas transgenic plants with antisense construct delayed the time of flowering. This result strongly suggests that 34B2T7 may be encoded by FLD gene. Further DNA sequencing indicated that gene encoded 34B2T7 containing 4 exons and three introns. A protein containing 335 amino acids long deduced from the nucleotide sequence of 34B2T7 shows 40% identity and 60% similarity to basic chitinase gene from several other plant species (rice, potato and wheat). The constructs expressed the product of 34B2T7 was constructed for further analysis of the function of protein.
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