利用報導基因融合方法研究FIN219基因表現的調控機制

Investigation of FIN219 gene expression by promoter fusion assay

鍾淑香(Shu-Hsing Chung)

指導教授 ： 謝旭亮

國立臺灣大學/生命科學院/植物科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01482

摘要

fin219 ( far-red insensitive 219)突變體乃是經由篩選cop1的基因外抑制體( extragenic suppressor )而來。此突變體乃是因位於啟動子區域DNA序列不正常的甲基化，導致在遠紅光下具有比野生型長的下胚軸外表型。FIN219屬於GH3-like基因家族成員之一，可受Auxin誘導而快速地表現，且參與光敏素A所導引的訊息傳遞。然而它在植物體中所扮演的角色及在光訊息傳遞中的功能尚不十分了解。因此，本論文利用報告基因( reporter gene )-GUS的協助，研究FIN219啟動子( promoter )的活性，以探討FIN219基因的表現及其調控的機制。
利用含有FIN219啟動子與GUS轉錄融合的T2轉殖株，針對不同生長階段在白光下的GUS染色分析，發現FIN219基因強烈表現在萌發中的種子、子葉的出水孔( hydathode )、毛茸( trichomes )基部及側根分叉點、花柱頭、花粉粒、及種子柄。由GUS的染色型態顯示FIN219基因表現的位置和植物荷爾蒙Auxin產生的地點非常一致，此結果與先前的研究FIN219可受Auxin誘導的特性吻合。
此外，我們亦利用轉殖株含有P219::GUS的融合質體來探討FIN219基因受光調控的表現機制。在light-switch的實驗結果顯示，轉殖株由黑暗轉移至遠紅光後其GUS活性變弱，相對於黑暗四天的轉殖株其GUS仍明顯的表現在子葉，推測遠紅光會抑制FIN219基因的表現。此外，在light-switch的實驗中我們利用由黑暗轉移至遠紅光的同時加入不同荷爾蒙，目的為檢測FIN219基因是否會受荷爾蒙的誘導，以及與遠紅光之間交感的調控。結果顯示，由黑暗轉移至遠紅光兩天後，原本GUS檢測不出的情形在加入荷爾蒙NAA、MeJA、JA誘導後，GUS染色明顯變強(ABA的誘導不明顯)，推測FIN219基因受遠紅光抑制的現象可以被荷爾蒙回復，似乎說明光和荷爾蒙間有某些的交感作用。
除此之外，含有FIN219 啟動子，coding region與GUS融合的野生型阿拉伯芥轉殖株，在遠紅光下具有hypersensitive的外表型(即下胚軸比野生型短)。並且它的成株具有部分不稔的現象。此結果與FIN219大量表現體在遠紅光或白光下的外表型一致。另外以北方點墨法(Northern blot )和西方點墨法( Western blot )分析這些轉殖株內生的FIN219基因及其蛋白質的表現量，結果發現轉殖株中內生FIN219基因的mRNA有兩個大小不同的條帶，而在野生型阿拉伯芥及fin219突變體中只偵測到一個條帶；而且轉殖株內的融合蛋白比內生的FIN219蛋白質降低很多。暗示轉殖基因受到轉譯或後轉譯層次的調控，造成融合蛋白的含量大大地降低，以致無法有效率的抑制fin219突變體的外表型。

關鍵字

光訊息傳遞

並列摘要

fin219 (far-red insensitive 219) mutant is derived from the screening of extragenic suppressors of the cop1-6 mutant in Arabidopsis. That abnormal DNA methylation was found in the promoter region of the fin219 mutant results in longer hypocotyl phenotype in far-red (FR) light than that in wild type. FIN219 belongs to a GH3-like gene family, is induced rapidly by auxin, and is involved in phytochrome A-mediated light signaling. However, its actual function and role in plant development and light signal transductions are not clear yet. So, the objective of my thesis is to study the promoter activity, gene expression patterns and possible regulatory mechanism of the FIN219 gene by transcriptional fusion with GUS.
According to the results of GUS staining carried out on different developmental stages of the T2 transgenic plants containing the P219::GUS fusion construct, FIN219 gene was highly expressed in the germinating seeds, the hydathod of cotyledons, the base of trichomes, the emerging point of lateral roots, and reproductive tissues, especially in the stigma, pollens as well as the funiculus. All these GUS staining patterns indicated that FIN219 gene expression was highly correlated to the site of auxin production and also consistent with the result reported previously that FIN219 transcripts were induced by auxin.
Besides, we also utilized the transgenic plants containing the P219::GUS fusion construct to study regulatory mechanisms of FIN219 expression regulated by light. The result from light switch experiments indicated that GUS activity was much reduced after transferring 2 day-old of dark-grown seedlings to FR light, when compared to that of dark-grown seedlings, implying that FR seems to inhibit FIN219 gene expression. Furthermore, this reduction of GUS activity in FR can be reversed by the addition of auxin, jasmonate or methyl jasmonate, suggesting that FIN219 may play an important role to integrate the cross talk between light and hormone signalings.
In addition, the transgenic plants containing the promoter and FIN219 coding region fused with GUS in wild type background exhibit a hypersensitive phenotype in cFR and their adult plants also show partial sterility, which was consistent with the result of FIN219 overexpressor in cFR and white light conditions. On the other hand, when we investigated the endogenous FIN219 expression in these transgenic plants by Western and Northern blot analyses, it was found that there were two endogenous FIN219 transcripts in transgenic plants, but only one in wild type and fin219 mutant. Further, FIN219-GUS fusion protein in transgenic plants was greatly reduced compared with the endogenous FIN219 protein, suggesting that the transgene expression was regulated at translational or posttranslational level, which resulted in the reduction of FIN219 fusion protein and then failed to rescue the fin219 mutant efficiently.

並列關鍵字

FIN219

參考文獻

Ahmad, M., and Cashmore, A.R. (1993). HY4 gene of A. thaliana encodes a protein with the characteristics of a blue-light photoreceptor. Nature 366, 162-166.

Ahmad, M., Lin, C., and Cashmore, A.R. (1995). Mutations throughout an Arabidopsis blue light photoreceptor impair blue-light-responsive anthocyanin and inhibition of hypocotyls elongation. Plant J. 8, 653-658

Anderson, M.B., Folta, K., Warpeha, K.M., Gibbons, J., Gao, J., and Kaufman, L.S. (1999). Blue light-directed destabilization of the pea Lhcb1*4 transcript depends on sequences within the 5' untranslated region. Plant Cell. 11, 1579-1590.

Ang, L.H., Chattopadhyay, S., Wei, N., Oyama, T., Okada, K., Batschauer, A., and Deng, X.W. (1998). Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Mol. Cell 1, 213–222.

Berry-Lowe, S.L., and Meagher, R.B. (1985). Transcriptional regulation of gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean tissue is linked to the phytochrome response. Mol. Cell. Biol. 5, 1910-1917.