葵百合LsGRP1蛋白質於葉片中之誘導表現及定位研究

Study on induced expression and localization of LsGRP1 in the leaves of Lilium cv. Star Grazer

姚先玟(Hsian-Wen Yao)

指導教授 ： 陳昭瑩

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00029

摘要

為探討LsGRP1 (Lilium ‘Star Gazer’glycine-rich protein 1)蛋白質在葵百合葉片之表現，將LsGRP1之cDNA構築於大腸桿菌蛋白質表現系統之載體上，進行LsGRP1表現與純化，發現細菌表現之LsGRP1蛋白質在電泳膠體上有偏移及部分C端胺基酸缺失的現象。進一步利用所得之LsGRP1製作多元抗體，得到含有LsGRP1抗體之兔子血清。使用LsGRP1抗體血清偵測，進行經水楊酸處理之葵百合葉片蛋白質萃取條件之測試，結果顯示利用非離子界面活性劑Triton X-100及還原劑硫氫乙醇(2-mercaptoethanol)萃取所得之蛋白質樣品中可偵測到較高量之38.2 kDa蛋白質訊號，而以高溫高鹼性處理及添加聚乙烯聚吡咯烷酮(polyvinylpolypyrrolidone)成分時可以偵測到約66 kDa的蛋白質訊號，又以高溫高鹼性處理所得66 kDa訊號較強。進一歩針對不同處理之葵百合葉片進行蛋白質萃取、電泳分析及偵測，所有處理皆可穩定地偵測到38.2 kDa蛋白質訊號，且於百合灰黴病菌感染之葉片蛋白質樣品中可偵測到最強的訊號，然而僅有在系統性誘導之系統葉蛋白質樣品中可偵測到66 kDa蛋白質訊號，推測LsGRP1可以巨大複合產物或是不同後轉譯修飾形式存在於植物中，於系統性誘導之系統葉中可以66 kDa蛋白質形式存在。此外，利用抗體偵測葵百合原生質體蛋白質表現以及觀察重組螢光蛋白質標記，探討LsGRP1蛋白質於細胞層次的累積位置，結果顯示於受到水楊酸刺激之原生質體細胞內與細胞外所得之蛋白質樣品中皆可偵測到66 kDa的蛋白質訊號，且隨著處理時間原生質體細胞外所得蛋白質訊號有增加的現象；進一步利用共軛焦顯微鏡(confocol)觀察綠色螢光蛋白質(green fluorescent protein, GFP)標記，結果證明具有LsGRP1訊息序列之重組綠色螢光蛋白質累積於細胞膜內側，推測葵百合葉片中之LsGRP1可能存在於細胞外質或細胞壁。

關鍵字

葵百合 ； LsGRP1 ； 多元抗體 ； 葵百合原生質體 ； 綠色螢光蛋白質

並列摘要

In order to study expression of LsGRP1 protein in Lilum ‘Star Gazer’ leaves, LsGRP1 cDNA was constructed in protein expression vector of Escherichia coli. LsGRP1 expression and purification were carried on for preparation of LsGRP1 polyclonal antibody. Western blot analysis detected LsGRP1 as a protein of 38.2 kDa following extraction with non-ionic detergent Triton X-100 and reductant β-mercaptoethanol. A protein of 66 kDa was detected following extraction with hot water-NaOH and polyvinylpolypyrrolidone. Extracts of Lilum ‘Star Gazer’ leaves in different treatments were stably detected the protein of 38.2 kDa, but the protein of 66 kDa was only detected in the protein samples of systemic leaves, indicating that posttranslational modification(s) or large mixture composition of LsGRP1 might occur. In addition, the protein of 66 kDa could be detected in SA-treated protoplasts, and protein signal changes from the cell to culture supernatant as the time increased. Confocol microscopy study indicated that LsGRP1-GFP fusion protein was not present evenly in the protoplast cytoplasm, but accumulated inside the cell membrane. Thus, LsGRP1 may exist in the extracellular matrix or cell wall in Lilum ‘Star Gazer’ leaves was presumed.

並列關鍵字

Lilum ‘Star Gazer’ ； LsGRP1 ； polyclonal antibody ； transient expression ； protoplast ； GFP fusion protein ； confocol microscopy

參考文獻

彭宣傑. 2006. 利用農桿菌注入法探討葵百合LsGRP1與水楊酸誘導抗病之相關性. 56頁。

Alba, M. N., Culianez-Macia, F. A., Goday, A., Freire, M. A., Nadal, B., and Pages, M. 1994. The maize RNA-binding protein, MA16, is a nucleolar protein located in the dense fibrillar component. Plant J. 6:825-834.

Aneeta, S. M. N., Tuteja, N., and Kumar, S. S. 2002. Salinity- and ABA-induced up-regulation and light-mediated modulation of mRNA encoding glycine-rich RNA-binding protein from Sorghum bicolor. Biochem. Biophys. Res. Commun. 296:1063-1068.

Berglund, G. I., Garlsson, G. H., Smith, A. T., Szoke, H., Henriksen, A., and Hajdu, J. 2002. The catalytic pathway of horseradish peroxidase at high resolution. Nature 417:463-468.

Birnboim, H. C., and Daly. J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.