水稻細胞主要分泌性蛋白質（Osm10）的鑑定與分析研究

Identification and characterization of a major extracellular protein (Osm10) secreted by suspension-cultured rice cells

周坤(Kun Chou)

指導教授 ： 王玉麒

國立臺灣師範大學/理學院/生命科學研究所/碩士(2012年)

摘要

水稻細胞在懸浮培養條件下會分泌多種蛋白質至培養液中，其中有一分子量約10 kDa的蛋白質 (簡稱Osm10) 的含量豐富，約佔全部分泌蛋白質總量的30%，本研究的目的即在鑑定生合成此蛋白質的基因與探討其表現特性。
為能鑑定Osm10的生合成基因，本研究首先利用二維膠體電泳將水稻細胞的分泌性蛋白質進行分離，再經蛋白質體學的方法分析後，得知Osm10主要包含三種蛋白質，命名為Osm10-I、Osm10-II及Osm10-III，皆屬第一群脂質傳送蛋白 (lipid transfer protein 1, LTP1) 的成員。Osm10-I與Osm10-II之間胺基酸序列相同度達96%，而Osm10-III和另兩個Osm10成員差異較大，胺基酸序列相同度都只有39%。立體結構模擬的結果顯示Osm10-I及Osm10-II屬於典型的LTP1，保守性序列D71、R72及Y107位於疏水性凹陷區域的開口周圍；Osm10-III則屬於特殊的LTP1，由第一與第三個α-helix圍成疏水性凹陷區域的開口，此立體結構的差異可能使Osm10-III具有與Osm10-I、Osm10-II不同的生理功能。若將Osm10與其他已知功能的LTP比較，Osm10-I及Osm10-II的胺基酸序列與大多數LTP的相同度約為50%，而Osm10-III與這些LTP的相同度則約為40%；Osm10-I及Osm10-II的立體結構最接近具抗真菌活性的LTP (ABO28527)，而Osm10-III的立體結構最接近與花粉管生長有關的LTP (Q9SW93)。利用資料庫分析Osm10基因的啟動子序列，結果顯示其序列可能具有種子專一性表現以及受多種植物激素與生物及非生物逆境調控的特徵。
本研究發現不僅是多種栽培稻，五種野生稻（O. alta、O. glaberrima、O. mtipogon、O. nivara、TWR-6）的細胞也同樣擁有Osm10基因且表現模式與栽培稻細胞一致，顯示Osm10可能在稻屬植物間具有特殊的生理功能。然而在本研究的分析條件之下，除了懸浮培養細胞的培養基中之外，水稻的種子、幼苗、成熟植株、花及發育中的種子都沒有發現有Osm10的表現，顯示Osm10可能是懸浮培養細胞專一性表現的蛋白質，或是必須使用更靈敏地分析方法才能確認Osm10的表現部位與表現時序特徵。本研究也使用與抗病或逆境相關的植物激素（SA、MeJA與ABA）來處理水稻細胞，結果並未觀察到顯著的差異變化。取水稻細胞的分泌性蛋白對四種水稻病原真菌（Rhizoctonia solani、Fusarium moniliforme、Bipolaris oryzae和Sarocladium oryzae）進行in vitro的抗真菌實驗，結果真菌的生長並不受抑制，可能代表Osm10不具備抑制受測病原真菌生長的活性。
本研究發現Osm10基因具備在懸浮培養的水稻細胞中專一且大量表現的特性，其啟動子及分泌調控序列可能具有應用於以懸浮培養的水稻細胞作為生物反應器，來生產高附加價值的外源重組蛋白質的潛力，除了可提升植物基因工程的應用之外，亦可避免基因改造植物對生態環境造成衝擊的疑慮。

關鍵字

水稻 ； 懸浮培養細胞 ； 脂質傳送蛋白 ； 分子模擬

並列摘要

Suspension-cultured rice cells secrete various proteins into the culturing medium, and over 30% of these secretory proteins were found to be a protein of 10 kDa in molecular mass (hereafter referred to as Osm10 protein). The aims of this research are to identify the encoding gene and to investigate the plausible functions and expression pattern of Osm10 in rice plants.
To identify Osm10 gene, Osm10 was first purified from 2-D gel electrophoresis, and analyzed using proteomic analytical methods. The results showed that Osm10 indeed consisted of 3 distinct proteins, designated as Osm10-I, Osm10-II and Osm10-III, and all of which are LTP1 proteins. Among these three Osm10 proteins, Osm10-I and Osm10-II share 96% amino acid sequence similarity, while Osm10-III only exhibits 39% sequence identity to the other two. Besides, 3D structural simulation pointed out that Osm10-I and Osm10-II are both typical LTP1s according to their conserved amino acid residues (D71, R72 and Y107) locating around the opening of hydrophobic cavity, while Osm10-III is an atypical LTP1 due to its opening of hydrophobic cavity surrounded by the 1st and 3rd α-helix. Structural difference of Osm10-III may contribute to a unique protein function other than common LTP1s. Sequences comparison with well studied LTPs also indicated that, Osm10-I and Osm10-II are about 50% identical to most of them, while Osm10-III shows only 40% identity. Structures of Osm10-I and Osm10-II resemble LTP with antifungal activity (ABO28527), on the other hand, Osm10-III are most like LTP relating pollen tube growth (Q9SW93). Analysis of Osm10 genes promoters revealed that their expression may be seed-specific and are regulated by plant hormones, biotic and abiotic stresses.
We found out that not only cultivated rice but also 5 species of wild rice (O. alta, O. glaberrima, O. mtipogon, O. nivara and TWR-6) cells highly express Osm10, which revealed that Osm10 proteins may play an
important role in Oryza genus. Nevertheless, Osm10 proteins were only detected in cultured cell medium, not in seeds, intact seedlings, flowers and immature seeds. Treatment with pathogen or stress resistance hormones (SA, MeJA and ABA) didn’t alter Osm10 expression. Secretory proteins of rice cells were also tested their anti-fungal activity against 4 fungal phytopathogens (Rhizoctonia solani, Fusarium moniliforme, Bipolaris oryzae and Sarocladium oryzae), but the growth of all the fungi being tested were not inhibited, indicating that Osm10 may not possess in vitro antifungal activity against the 4 fungi tested in the research. These results, overall, indicates that expression of Osm10 may be suspension-cell-specific.
The suspension-cell-specific and highly-expressing feature of Osm10 gene suggest a highly profitable application of its promoter in production of heterologous recombinant protein within rice cell culture system, which can also be used to avoid the doubt of gene-modified crops in environmental aspect.

並列關鍵字

rice ； suspension cell ； lipid transfer protein ； molecular modeling

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