綠竹 BohLOL1 基因之選殖與分析

Cloning and Analysis of LSD-one-like 1 Gene from Bambusa oldhamii

王人雙(Jen-Shuang Wang)

指導教授 ： 王愛玉

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10729

摘要

BohLOL1 (Bambusa oldhami LSD-One-Like 1) 基因轉譯出的蛋白質具有 DNA 結合能力且存在於細胞核中，其胺基酸序列具有三個 zf-LSD1 (zinc finger-Lesions Simulating Disease resistance 1) domains。在過去研究中發現於快速生長時期的綠竹筍中，BohLOL1 mRNA 含量大幅提升，此外以 auxins、cytokinins 或病原菌處理竹苗後，mRNA 含量亦會大幅提升。本研究以 PCR選殖出 BohLOL1 的genomic DNA，將其與 cDNA 序列比對後，顯示 BohLOL1 基因結構具有 5 個 exons、4 個 introns。然而，將 BohLOL1 基因與其他植物 LSD 及 LOL 相關基因之結構比較後，推測 BohLOL1 可能在轉譯起始點上游仍有一個 exon 存在。此外，利用 genome walking 法選殖 BohLOL1 轉譯起始點之上游序列，並預測此序列上之順式調控因子 (cis-elements)，發現許多與植物荷爾蒙或逆境相關的因子，且在孟宗竹 (Phyllostachys edulis) 同源基因 PH01001063G0080 (稱之為 PeLOL1) 的基因調控序列中，也有預測到相同的順式調控因子。BohLOL1 與 PeLOL1在核苷酸及胺基酸序列之同質性高，兩者的基因結構及調控序列上之順式調控因子亦具保留性，顯示 BohLOL1 與 PeLOL1 為直系同源基因，並且在植物生長與防禦的過程中具有相似的功能。

關鍵字

竹生長發育 ； 綠竹 ； 抗病性 ； 順式調控因子

並列摘要

BohLOL1 (Bambusa oldhami LSD-One-Like 1) encodes a nuclear DNA-binding protein containing three zf-LSD1 (zinc finger-Lesions Stimulating Disease resistance 1) domains. This gene has been shown to be upregulated in bamboo shoots with higher rates of culm elongation, and also upregulated by auxins, cytokinin, and pathogen infection. In this study, BohLOL1 genomic DNA was cloned using PCR. Comparing the genomic DNA sequence of BohLOL1 with its cDNA sequence revealed that BohLOL1 gene had 5 exons and 4 introns. However, the result of sequence alignment of BohLOL1 with LSD1- and LOL1- related genes from other plants suggested that BohLOL1 had another exon upstream the translation start site. Genome walking was performed to clone the upstream sequence. Many kinds of putative regulatory cis-elements were found in the sequence upstream the predicted transcription initiation site. Some cis-elements were related to pytohormones or stresses, and these cis-elements could also be found in the promoter region of a homologous gene (PH01001063G0080, designated PeLOL1) in moso bamboo (Phyllostachys edulis). BohLOL1 and PeLOL1 show high nucleotide and amino acid sequence identities. Conservation of structural gene sequence and cis-elements in their regulatory regions suggested that BohLOL1 and PeLOL1 were orthologs and had similar biological functions in plant growth or disease resistance.

並列關鍵字

BohLOL1 (Bambusa oldhami LSD-One-Like 1) ； bamboo growth ； disease resistance ； cis-elements

參考文獻

Alexandrov, N.N., Brover, V.V., Freidin, S., Troukhan, M.E., Tatarinova, T.V., Zhang, H., Swaller, T.J., Lu, Y.P., Bouck, J., Flavell, R.B., Feldmann, K.A. (2009) Insights into corn genes derived from large-scale cDNA sequencing. Plant Mol Biol, 69(1-2), 179-194.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (1987) Current Protocols in Molecular Biology, John Wiley Sons, Inc, New York.

Aviv, D.H., Rusterucci, C., Holt, B.F., Dietrich, R.A., Parker, J.E., Dangl, J.L. (2002) Runaway cell death, but not basal disease resistance, in lsd1 is SA‐and NIM1NPR1‐dependent. The Plant Journal, 29(3), 381-391.

Bhatti, K.B., Ahmed, N., Shah, A., Iqbal, M., Iqbal, T., Jiahe, W. (2011) Transgenic tobacco with rice zinc-finger gene OsLOL2 exhibits an enhanced resistance against bacterial-wilt. Australasian Plant Pathology, 40, 133-140.

Bhatti, K.H., Xu, C., Wu, J., He, C. (2008) Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. tabaci. Progress in Natural Science, 18(7), 807-812.