Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97561
標題: 利用T-DNA插入突變體探討水稻基因之功能-抗病能力異常突變體M23454基因功能之研究
Functional characterization of a pathogen susceptible T-DNA insertion mutant- M23454
作者: 許揚
Yang Hsu
關鍵字: 水稻;突變株;轉錄因子;稻熱病;乾旱;Rice;T-DNA insertion mutant;Pathogen-related gene
引用: 李彥璇 (2006) 利用T-DNA插入突變體探討水稻基因之功能-抗病能力異常突變體M0023454及穀粒發育異常突變體M0039314之特性及基因表現 沈盟倪 (2008) 利用T-DNA插入突變體探討水稻基因之功能-抗病能力異常突變體M0023454基因功能之研究 余承駿 (2013) 水稻抗病能力異常T-DNA突變體M0023454及高分蘗M0111350突變株之基因功能探討 Arciga-Reyes, L., Wootton, L., Kieffer, M., and Davies, B. (2006). UPF1 is required for nonsense-mediated mRNA decay (NMD) and RNAi in Arabidopsis. Plant J 47, 480-489. Baldrich, P., Campo, S., Wu, M.T., Liu, T.T., Hsing, Y.I., and San Segundo, B. (2015). MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors. RNA Biol 12, 847-863. Banerjee, A., and Roychoudhury, A. (2015). WRKY proteins: signaling and regulation of expression during abiotic stress responses. ScientificWorldJournal 2015, 807560. Bowman, J.L., Smyth, D.R., and Meyerowitz, E.M. (1989). Genes directing flower development in Arabidopsis. Plant Cell 1, 37-52. Chorev, M., and Carmel, L. (2012). The function of introns. Front Genet 3, 55. 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摘要: 
M23454為台灣水稻資料庫 (TRIM database) 所建立的T-DNA插入水稻突變株,其利用T-DNA上構築的CaMV35S增強子,活化了插入位附近的454-10基因,在外表性狀上M23454相較於WT (TNG67) 呈現矮株易感病的現象,並可以在葉片上觀察到明顯的稻熱病病斑,推測454-10基因可能在植物免疫調控上扮演某種重要角色。實驗室利用植物基因轉殖的技術,構築以玉米ubiquitin啟動子大量表現454-10 (cDNA) 基因之水稻突變株 (Ubi::454-10/cDNA),推測可能造成植株抵抗病菌能力產生變異,然而經過幾季田間植株的觀察,並沒辦法觀察到與M23454相似的外表性狀。另外為了驗證造成M23454外表性狀之原因是否是因為植株本身內生性的因子,又或是受外界環境壓力所誘導造成,將M23454分別種植於溫室相對無病原之環境,對比種植於田野中的植株可發現外在環境對植株的影響大於內生性的原因。
利用NCBI及系統演化樹的分析,454-10的蛋白內含有一個AP2 domain,歸類上屬於AP2/EREBP (APETALA2/ethylene response factor) 家族ERF subgroup的transcription factor,主要參與調控生物性逆境的反應,在前人的研究中也發現病機制相關的植物賀爾蒙中,ethylene,jasmonic acid和salicylic acid會誘導454-10的表現,而ABA卻會抑制454-10的表現,證明454-10確實參與了逆境訊息傳遞的調控。
454-10基因包含了2個Exon和1個Intron,為了排除基因可能存在不同的剪接修飾,導致所調出cDNA片段無法回復突變株的外表性狀,於是設計新的PCR引子調出全長的454-10 (gDNA) 並利用同樣基因轉殖技術將此片段大量表現,得到轉殖株 Ubi::454-10/gDNA,結果發現在外表性狀上此轉殖株確實恢復了與M23454相同之感病性狀。另外取90 DAI植株抽RNA進行RT-PCR分析,發現454-10 (gDNA) 的植株在RNA表現上相較於454-10 (cDNA) 的植株,出現一個明顯且片段較大的序列,將此片段純化送定序後,發現其為未被剪切的Intron序列,細部去推測是否是因為這段intron的序列導致植株外表出現差異,我們在intron的序列中發現一個miRNA5819的預測結合位,然而其miRNA功能尚未被研究。在過去的研究中已經證實miRNA參與了水稻對於逆境的免疫反應,但是大多作用於編碼區域,關於miRNA與intron之間的交互作用研究並不多。是否miR5819會去結合上454-10基因之intron導致植株外表出現變異仍有待後續研究。

A rice T-DNA insertion mutant M23454 revealed susceptible to pathogens and prone to form leaf lesions during the transition from vegetative growth stage to reproductive stage. This mutant has its T-DNA inserted in the rice chromosome #4 within the BAC clone OSJNBa0039C07 region. All predicted genes flanked to the insertion site within this BAC clone region were analyzed. The flanking gene named 454-10 was the only one significantly activated in the mutant; however, transgenic line over-expressing a full-length cDNA of 454-10 did not recapitulate the phenotype of M23454. An allelic mutant that has its T-DNA inserted next to 454-10 gene and activate expression of 454-10 showed similar phenotype of M23454. The observation in allelic mutant further assured the positive correlation between the phenotype of M23454 and its T-DNA insertion event.
The gene 454-10 with locus name LOC_Os04g32620 was classified as the B-1 subgroup of ERF family, containing 11 putative DNA binding sites, and was predicted as a key gene response to stress and abiotic stimuli. A further attempt to over-expressing a gDNA construct containing 5'UTR and intron of 454-10 recapitulated the phenotype of M023454. the different results between cDNA and gDNA construct indicate that the sequences other than the coding region also play important roles in regulating the function of 454-10. RT-PCR analysis of the 454-10/gDNA transgenic lines showed, in addition to the 454-10 RNA transcript, an intron-containing RNA transcript was observed. This observation suggests that the sequences of intron might play important roles in regulating the function of 454-10. Through online database searching, a target site of miR5819 in the middle sequence of the intron was found. MicroRNAs (miRNA) have been considered as one of the important small RNAs element that might target to the noncoding region to regulate gene expression to combat various plant stresses. Whether the pathogen susceptible phenotype of M23454 is caused by the overexpression of 454-10-intron and/or the interaction of intron with miR5819 require further investigations.
URI: http://hdl.handle.net/11455/97561
Rights: 同意授權瀏覽/列印電子全文服務,2022-01-22起公開。
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