調節GA2ox基因表現之可能轉錄因子探討及具高分蘖數T-DNA突變株M69217之研究

白育旻

doi:10.6845/NCHU.2015.01105

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調節GA2ox基因表現之可能轉錄因子探討及具高分蘖數T-DNA突變株M69217之研究

Searching for transcription factors involved in regulating expression of GA2ox genes and characterization of a high tiller number T-DNA insertion mutant M69217

白育旻 , Masters　　Advisor：陳良築　　

繁體中文 DOI： 10.6845/NCHU.2015.01105

激勃素；轉錄因子；分蘖； GA ； DOF ； tillering

Abstract │ Reference (58) │ Altmetrics

Abstract 〈TOP〉

GA (gibberellic acid)為一個參與植物發育之賀爾蒙，然而過多的GA含量會導致植株易倒伏，缺乏GA時會使植株矮化。GA2-oxidases為一個可以將活性GA或GA前驅物代謝之酵素，目前已知水稻GA2ox由10個基因所編碼，分別為 OsGA2ox1-10，，其中OsGA2ox10可能為pseudo gene，目前仍未有研究探討GA2ox之轉錄調節機制，本研究之目的為尋找參與GA2ox基因表現之轉錄因子。經生物資訊分析後發現OsGA2oxs啟動子皆具有DOF (DNA binding with one finger)之結合位，分析水稻33個OsDOF之DOF domain進行演化樹分群並且根據分群找出OsDOFACT (M51680 and M53794)、OsDOF7ACT (M63981)、OsDOF10ACT (M45005)、OsDOF10KNO (M78151)、OsDOF24KNO (M61519)及OsDOF25ACT (M66458)等7個突變株。經田間觀察，7個突變株中只有2個有矮化性狀，其編號分別為OsDOF1ACT (M53794)與OsDOF24KNO (M61519)。分析M53794之DOF1與M61519之DOF24之基因表現，只有在M53794有偵測到目標基因OsDOF1被活化，OsDOF24於M61519表現與在TNG67無明顯差異。
分析OsGA2ox基因於M53794與M61519之表現，不僅與TNG67無明顯表現差異而且不具一致性，推測OsDOF1與OsDOF24並不調控OsGA2ox轉錄調控而且OsGA2ox基因表現不一致是因環境因子導致。
	T-DNA突變株M69217為具有矮株與多分蘖外表性狀，且鄰近基因CM (Chorismate mutase) and CCR (Cinnamoyl-CoA reductase)皆有被活化。然而大量表現CM或CCR於水稻後的外表形與M69217並不相同，本研究將進一步探討其外表性狀及導致其外表形之基因。分析M69217的microarray研究得知， GCN5-related N-acetyltransferase (GNAT)位於第二號染色體，而且為M69217 T-DNA插入位之鄰近基因，經分析，GNAT於M69217有偵測到活化現象，然而Ubi::GNAT轉殖株目前仍無法成功獲得。M111350與M69217一樣具高分蘖數，前人研究指出M111350之高分蘖數是miR156b活化所導致，經NCBI比對，M69217 T-DNA插入位位於miR156d DNA序列，然而分析miR156d於M69217之表現，結果卻顯示M69217之miR156d表現與TNG67無明顯差異，但受miR156抑制的SQUAMOSA Promoter-Binding-Like Transcription Factors 16 (SPL16)卻呈現低表現量。未來將對miR156d進行轉基因做大量表現並探討miR156d與
M69217具高分蘖數之關聯性。

Parallel Abstract 〈TOP〉

GA (gibberellic acids) is one of the plant hormones involved in plants development, excessive GA cause plant lodging and lack of GA cause plant dwarf. GA2-oxidases are enzymes catalyze bioactive GA or GA precursors to inactive GAs and to regulate GA homeostasis in plants. There are nine GA2-oxidase genes in rice (OsGA2ox1–9) and how these gene expressions are regulated temporally and spatially in rice plant have not yet been characterized. This study intended to search for transcription factors involved in regulating the expression of OsGA2ox genes. After bioinformatics analysis, promoter sequences of most OsGA2ox genes contain DOF (DNA binding with one finger) transcription factor binding site. Phylogenetic analysis of 33 OsDOFs and their possible involvement in GA regulation based on previous studies, totally 7 T-DNA insertion mutants, including OsDOFACT (M51680 and M53794)、OsDOF7ACT (M63981)、OsDOF10ACT (M45005)、OsDOF10KNO (M78151)、OsDOF24KNO (M61519) and OsDOF25ACT (M66458), from TRIM library were collected. After field observation, mutant M53794 (for OsDOF1 target gene) and mutant M61519 (for OsDOF24 target gene) revealed slightly dwarf phenotype and were further characterized. The RNA expression of two target genes, OsDOF1 and OsDOF24, were analyzed by RT-PCR. Gene of OsDOF1 showed increased expression in M53794 and DOF24 in M61519 showed the same expression level as those of TNG67. Expression of OsGA2ox genes in these two mutants were analyzed and neither significant nor consistent levels of expression for OsGA2ox genes was observed between mutant and TNG67, suggested that the expression of OsGA2ox genes were not regulated by OsDOF1 or OsDOF24 and the inconsistent expression of OsGA2ox genes might cause by the environment factors.
Mutant M69217 showed severe dwarf and high tiller number and its flanking genes CM (Chorismate mutase) and CCR (Cinnamoyl-CoA reductase) were activated. However overexpression of CM and/or CCR genes could not recapitulate the phenotype of M69217. In this study, further characterization of this mutant and identification of genes responsible for its phenotype was investigated. Further analysis showed another gene, GNAT (GCN5-related N-acetyltransferase), located near the T-DNA insertion site was also activated. Overexpressing GNAT in transgenic rice to investigate its effect was underway. Another T-DNA insertion mutant M111350 revealed the same phenotype as that of M69217 and results suggested that the unstoppable tillering might cause by activation of miR156b. It was interesting to realize that another miR156d gene was located near the insertion site as well. However, the expression of miR156d in M69217 was no significant different from that in TNG67, but the miR156d target gene SPL16 (SQUAMOSA Promoter-Binding-Like Transcription Factors) decreased in M69217. Overexpressing miR156d in transgenic rice was performed to investigate its effect on tillering.

Reference ( 58 ) 〈TOP〉

Achard, P., Gong, F., Cheminant, S., Alioua, M., Hedden, P., and Genschik, P. (2008). The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism. The Plant cell 20, 2117-2129.
連結：
Appleford, N.E., Evans, D.J., Lenton, J.R., Gaskin, P., Croker, S.J., Devos, K.M., Phillips, A.L., and Hedden, P. (2006). Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat. Planta 223, 568-582.
連結：
Bastiani, M.J., Harrelson, A.L., Snow, P.M., and Goodman, C.S. (1987). Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper. Cell 48, 745-755.
連結：
Borner, G.H., Lilley, K.S., Stevens, T.J., and Dupree, P. (2003). Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A proteomic and genomic analysis. Plant physiology 132, 568-577.
連結：
Chalfun-Junior, A., Mes, J.J., Mlynarova, L., Aarts, M.G., and Angenent, G.C. (2003). Low frequency of T-DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences. FEBS letters 555, 459-463.
連結：

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