阿拉伯芥atToc159 轉運蛋白基因的細胞專一性表現與領先內插子對基因表現的影響
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Date
2013-??-??
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國立臺灣師範大學生命科學學系
Department of Life Science, NTNU
Department of Life Science, NTNU
Abstract
的同源蛋白質，分成atToc159/atToc90 和atToc132/atToc120 兩大群。其中atToc159/atToc90被認為與辨識和運輸光合作用蛋白質有關，atToc132/atToc120 則可能負責辨識和運輸非光合作用蛋白。在阿拉伯芥生長發育的過程中，這些轉運蛋白的基因必須被適當的調控，否則色質體的發育與生理將因缺乏必需的蛋白質的輸入而被抑制。為了解atToc159 基因家族的表現機制，轉殖分別攜帶不同長度的基因上游調控序列的重組質體進入野生型阿拉伯芥，以獲得穩定轉殖的植株。分析這些轉殖植株中營養器官的GUS 酵素活性及mRNA 表現量，發現atToc159、atToc132/90 和atToc120 基因在七天齡的幼苗中分別有最高，次之及最低的表現量。此外，位於atToc120 和atToc90 的5'UTR 內的領先內插子序列會明顯增加其基因表現量。進一步探討光對內插子在基因表現上的影響，比較生長在光照和黑暗條件下的120PUI 和120P 轉殖株的GUS 表現量，我們發現黑暗會提高內插子效應。除了進行不同組織的基因表現之比較分析外，我們也進行了這些轉殖株中基因的細胞專一性表現的研究。令人驚訝的，在與atToc159 和atToc132 的比較下，atToc90 和atToc120 在子葉保衛細胞中基因的表現量明顯地要比葉肉細胞多，這顯示atToc90 和atToc120 在保衛細胞運輸蛋白質進入葉綠體上可能扮演一定的角色。這些研究結果支持葉綠體轉運蛋白atToc159 基因家族之基因的表現量在不同的組織和發育階段的差異，可能是受到不同的發育和環境的因子刺激所調節。
In Arabidopsis, four Toc159 homologues are reported and could be classified to two subgroups, atToc159/atToc90 and atToc132/atToc120, based on their sequence homology and substrate specificity. Therefore, expression of these translocon genes have to be regulated properly, otherwise the development and maintenance of plastids would be prohibited due to lack of essential protein import. In order to reveal the regulatory mechanism of atToc159 gene family members, transgenes containing various lengths of the upstream sequences of these translocon genes and GUS coding sequence were transferred to wild-type Arabidopsis. The steady accumulation of GUS expression from the vegetative tissues of these transformants was examined first. In general, atToc159, atToc132/atToc90, and atToc120 have relatively high, moderate, and low expression levels in various tissues of 7-day-old seeldings. Furthermore, the expression of atToc120 and atToc90 could be up-regulated by their corresponding leader intron sequence within 5' untranslated region (5' UTR) region. To further understand the intron effect of atToc120 expression was regulated by light, the GUS activity of 120PUI and 120P plants grown under light/dark cycle and continuous dark conditions were compared. Our data suggests that the intron sequence of atToc120 is more responsive to dark signal. In addition to tissue-specific expression, the cell-specific expression of these transgenic plants was also determined. Surprisedly, atToc90 and atToc120 had higher GUS activity in the guard cells than mesophyll cells of cotyledon when compared to atToc159 and atToc132. This indicates that atToc90 and atToc120 might play a more important role in regulating chloroplast protein import of guard cells. These data support that the spatial and temporal expression of atToc159 gene family members is regulated by different developmental and environmental stimuli.
In Arabidopsis, four Toc159 homologues are reported and could be classified to two subgroups, atToc159/atToc90 and atToc132/atToc120, based on their sequence homology and substrate specificity. Therefore, expression of these translocon genes have to be regulated properly, otherwise the development and maintenance of plastids would be prohibited due to lack of essential protein import. In order to reveal the regulatory mechanism of atToc159 gene family members, transgenes containing various lengths of the upstream sequences of these translocon genes and GUS coding sequence were transferred to wild-type Arabidopsis. The steady accumulation of GUS expression from the vegetative tissues of these transformants was examined first. In general, atToc159, atToc132/atToc90, and atToc120 have relatively high, moderate, and low expression levels in various tissues of 7-day-old seeldings. Furthermore, the expression of atToc120 and atToc90 could be up-regulated by their corresponding leader intron sequence within 5' untranslated region (5' UTR) region. To further understand the intron effect of atToc120 expression was regulated by light, the GUS activity of 120PUI and 120P plants grown under light/dark cycle and continuous dark conditions were compared. Our data suggests that the intron sequence of atToc120 is more responsive to dark signal. In addition to tissue-specific expression, the cell-specific expression of these transgenic plants was also determined. Surprisedly, atToc90 and atToc120 had higher GUS activity in the guard cells than mesophyll cells of cotyledon when compared to atToc159 and atToc132. This indicates that atToc90 and atToc120 might play a more important role in regulating chloroplast protein import of guard cells. These data support that the spatial and temporal expression of atToc159 gene family members is regulated by different developmental and environmental stimuli.