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標題: 阿拉伯芥中兩個EPF基因的特性分析 及功能性探討
Characterization and functional analysis of two EPF genes from Arabidopsis thaliana
作者: 呂官杰
Leu, Kuan-Chieh
關鍵字: EPF
出版社: 生物科技學研究所
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摘要: EPF (EPSP合成酶因子)基因是第一個植物中被鑑定出的C2H2型式存在的鋅指蛋白,並且EPF家族都含有特殊的保守序列CX2CX3FX5LX2HX3H。本研究對EPF家族中的兩個基因AtEPF1及AtEPF2進行選殖分析之工作。AtEPF1專一的表現在成熟的花朵中,而AtEPF2基因則表現在小苗、葉子,根、莖及花朵中。在啟動子分析實驗中,以AtEPF1基因的啟動子驅動GUS基因,GUS活性只在成熟的花藥及花粉中被偵測到;而以AtEPF2基因的啟動子驅動GUS基因下,GUS活性在小苗、根、莖、葉子、花粉中和果莢中被偵測到。此外,以35S啟動子異位表現AtEPF1基因或AtEPF2基因皆可得到使植株矮小化及造成葉子有近似脫水的性狀。進一步以「農桿菌注射」的方式在菸草中短暫表現AtEPF1基因及AtEPF2基因,結果亦都可以得到近似脫水的性狀。對AtEPF1或AtEPF2基因進行專一性之antisense,都無法得到明顯性狀,此點顯示此二基因的功能可能具有重疊性。進一步對T-DNA插入造成AtEPF1基因功能性缺失的植株進行觀察,發覺有明顯的不孕及花粉聚集成塊的性狀。綜合上述實驗數據,我們認為AtEPF1基因或許在花粉發育過程中專一的調控花粉的脫水步驟;而AtEPF2基因則為AtEPF1基因調控花粉脫水的同功能性基因。因此我們進行35S::AtEPF1基因轉基因植株的生物微晶片實驗分析,找到了一個受AtEPF1基因調控大量表現的基因,經由初步進行的實驗,推測該基因可能會對脫水情況有所反應或保護植株,因此大量表現此基因之植株對乾旱的逆境應會有較強的抵抗性。
EPF (EPSPS ((5-enolpyruvylshikimate-3-phosphate synthase)) Factor) was the first Cys2/His2 zinc finger protein identified from plants. The EPF family contains the conserve sequences CX2CX3FX5LX2HX3H. Two EPF genes AtEPF1 and AtEPF2 were cloned and characterized in this study. AtEPF1 is specifically expressed in mature flower whereas AtEPF2 is expressed in seedling, leaf, root, stem and flower. In AtEPF1::GUS plants, the GUS activity was specifically detected in anther and pollen of the mature flower. In AtEPF2::GUS plants, the GUS activity was detected in seedling, root, stem, leaf, pollens and silique. Ectopic expression of either AtEPF1 or AtEPF2 caused a similar phenotype by significantly reducing the plant size and producing curried and dehydrated leaves. Furthermore, transient expression of AtEPF1 or AtEPF2 in tobacco leaves by using “Agro-infiltration” produced similar phenotypes of dehydration for the leaves. Both AtEPF1 and AtEPF2 specific antisense mutant were phenotypically indistinguishable from wild type plants. However, the male sterility phenotype with congregated pollens was observed in the T-DNA insertion mutant line of AtEPF1. These results indicated that AtEPF1 and AtEPF2 are likely the homologues with redundant function and specifically regulate dehydration of the pollens during pollen development. Further microarray analysis for 35S::AtEPF1 plants identified a gene that significantly up-regulated by AtEPF1. The function of this gene may be in response to dehydration. Further functional analysis transgenic plants ectopically expressing this gene in response to dehydration stress should reveal it function.
其他識別: U0005-1908200922562500
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