具葉緣鋸齒性狀之阿拉伯芥T-DNA插入突變株TPR8之功能性基因研究

Funtional study of a T-DNA insertional mutant, TPR8, with serrated leaves in Arabidopsis thaliana

何盈儒(Yin-Ju Ho)

指導教授 ： 靳宗洛

國立臺灣大學/生命科學院/植物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10415

摘要

調控植物葉部形態的機制複雜，可受到許多因子的影響如極性生長，荷爾蒙的分佈，細胞的分化與排列等。本實驗以具有2x35S:Bar:SK的T-DNA片段隨機插入阿拉伯芥的基因組（genome），篩選具有葉部形態變異的突變株，探討受影響的基因功能及其與突變性狀間的關連性。目前本研究取得暫命名為TPR8之突變株，其外表性狀明顯地從第四片葉開始以及其後生長的新葉，較野生型具有明顯的鋸齒狀葉緣；且喪失頂芽優勢；延遲老化故生命週期比野生型長。以上的性狀，類似處理細胞分裂素(cytokinin)後所呈現的表型，因此推測TPR8中所影響的基因中，其功能可能與細胞分裂素的訊號傳遞路程有關。實驗證明由基因組中編號為At4g01245功能未知的基因，因T-DNA的插入而提高其基因表現量造成上述之性狀。目前對於At4g01245並無任何的相關研究可供參考，因此利用已知會受細胞分裂素活化表現的A型ARRs (type-A Arabidopsis response regulators)，來探討TPR8、At4g01245及細胞分裂素之相關性。在TPR8突變株以及在At4g01245大量表現後可重現TPR8外表型的突變株中，細胞分裂素訊息傳遞路徑中的負調控因子，包括ARR4, ARR5, ARR7 及ARR9 的表現量均下降，顯示At4g01245參與細胞分裂素訊息傳遞路徑。此外，TPR8突變株中的另一At4g01250基因，因T-DNA插入在其調控子上而抑制其表現，由於TPR8自交的子代中，可分離出少數的矮化植株，此性狀可在At4g01250 knock out自交株中重現，因此推測At4g01250的表現量可調節植物的生長及發育。

關鍵字

細胞分裂素 ； 鋸齒狀葉緣 ； 阿拉伯芥

並列摘要

The regulatory mechanism of leaf morphogenesis is complex and influenced by a lot of factors, such as the polar growth, the distribution of phytohormones, and the cell differentiation and so on. We used T-DNA containing 2x35S:Bar:SK to transform Arabidopsis for screening mutants with abnormal leaf phenotypes for further investigating the influences and the functions of genes that are correlated with the mutant phenotype. We focused on a gain-of-function mutant, TPR8, with the At4g01245 overexpressed, with leaves from the fourth leaf to the later sprouting leaves are more obviously serrated than the wild type. The TPR8 also share the longer life cycle, the losing of apical dominance and the serrated leaves, as the effect of plant treated with cytokinin. Thus far, the function of At4g01245 is still unclear, so some members of type-A ARRs family (type-A Arabidopsis response regulators) which activated by cytokinin and functioning as negative regulator were used to confirm the relationship between TPR8, At4g01245 and cytokinin signaling. The transcriptional levels of ARR4, ARR5, ARR7 and ARR9 were all decreased in the TPR8 and also in the At4g01245 overexpression lines. It suggests that At4g01245 may participate in down regulation of the negative regulators, to promote cytokinin responses involved in the regulation of cytokinin signaling pathway. Furthermore, the gene At4g01250 expressed in TPR8 mutants was down regulated as the T-DNA inserted in the promoter and could segregate some dwarf mutant in the progeny, as seen in the At4g01250 null mutant lines. It implied that the expression of At4g01250 is connected with growth and development in planta.

並列關鍵字

T-DNA ； cytokinin ； TPR8 ； Arabidopsis ； serrated

參考文獻

Altmann, T. (1998). Recent advances in brassinosteroid molecular genetics. Curr Opin Plant Biol 1, 378-383.

Autran, D., Jonak, C., Belcram, K., Beemster, G.T., Kronenberger, J., Grandjean, O., Inze, D., and Traas, J. (2002). Cell numbers and leaf development in Arabidopsis: a functional analysis of the STRUWWELPETER gene. EMBO J 21, 6036-6049.

Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992). New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20, 1195-1197.

Bechtold, N., Ellis, J., Pelletier, G. (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci 316, 1194-1199

Bongard-Pierce, D.K., Evans, M.M.S., and Poethig, R.S. (1996). Heteroblastic features of leaf anatomy in maize and their genetic regulation. Plant Sci. 157, 331-340.