水稻（Oryza sativa L.）Osm30分泌性蛋白質的生化及功能分析—一種第三型的幾丁質酶

Biochemical and Functional Characterization of Rice (Oryza sativa L.) Osm30 Secretory Protein-A Class III Chitinase

李嘉雯(Chia-Wen Li) ； 王玉麒(Yu-Chie Wang)

《Bio Formosa》 41卷1期 (2006/06) Pp. 31-43

https://doi.org/10.6248/BF.2006.41(1).05

摘要

本研究自水稻的懸浮細胞培養液中，分離出一分子量為30 kDa，且具有水解glycol chitin活性之蛋白質（Osm30）。經由蛋白質N端胺基酸微定序分析及基因選殖的結果顯示，Osm30為水稻的第III型幾丁質酶家族的一員。將Osm30基因轉形並表現於大腸桿菌時，可觀察到細菌生長被抑制的情形，推測Osm30可能具有溶菌酶的活性。在水稻細胞懸浮培養的過程中，Osm30的mRNA會因繼代培養而有暫時性增加表現的情形，但其在細胞內的蛋白質含量則相對穩定，而在培養液中，Osm30蛋白質則是隨著培養天數增加而持續累積，顯示Osm30蛋白質具有高度穩定性。當以四種病原真菌，包括：Rhizoctonia solani、Sarocladium oryzae、Gibberella fujikuroi 和Bipolaris oryzae，感染水稻細胞時，均會誘導增加Osm30的表現量，顯示Osm30可能在水稻的抗菌防禦機制中扮演相關角色。甲基茉莉酸（MeJA）、離層酸（ABA）和水楊酸（SA）為已知和植物防禦機制有關的生長調節劑，當以這些生長調節劑對水稻細胞進行處理時，MeJA與ABA可誘導Osm30的表現，但是SA的處理則不影響Osm30的表現。此外，我們也在Osm30基因的起動子區域發現與JA和ABA相關的轉錄調控位。根據上述資料，我們推測Osm30為一具有幾丁質酶和溶菌酶雙重酵素活性的蛋白質，在水稻細胞內平常只有基礎量的表現，但當病原菌入侵時，可能經由JA或／和ABA啟動的訊息傳遞途徑來激發Osm30的表現，以強化植物的防禦機制。

關鍵字

水稻 ； 幾丁質酶 ； 溶菌酶 ； 茉莉酸 ； 離層酸

並列摘要

A 30-kD protein secreted by suspension-cultured rice cells (Osm30) was identified with activity of cleaving glycol chitin. Furthermore, data of amino acid microsequencing and gene cloning confirmed that Osm30 is a member of rice Class III chitinase gene family. Over-expression of recombinant Osm30 in E. coli resulted in a retarded growth of the bacteria, suggesting Osm30 might possess lysozyme activity of the Famiy 18 glycosyl hydrolase. Upon transfer of rice cells to fresh culture medium, Osm30 transcripts display a transient increased expression pattern in rice cells. However, Osm30 protein maintains a constant level in rice cells and is constitutively secreted into the medium. These results suggest that Osm30 protein is highly stable in vivo and in vitro. Inoculation of any of the four fungal pathogens, including Rhizoctonia solani, Sarocladium oryzae, Gibberella fujikuroi, and Bipolaris oryzae can cause an elevation of the expression levels of Osm30 in rice cells, indicating that Osm30 may play a role in plant defense mechanism against fungal infection. Among the growth regulators that are known to be involved in plant defense mechanisms, MeJA and ABA are able to upregulate the Osm30 expression levels, but SA is not. In addition, there are several JA- and ABA-responsive cis-acting elements identified in the promoter regions of the Osm30 gene. In summary, Osm30 appears to be a protein with both chitinase and lysozyme activities. Suspension-cultured rice cells express Osm30 at a basal level prior to pathogen infection. Upon pathogen infection, rice cells raise the expression level of Osm30, probably via JA and/or ABA-initiated signal transductional pathway(s), to augment cellular defense mechanism.

並列關鍵字

Oryza sativa ； chitinase ； lysozyme ； jasmonic acid (JA) ； abscisic acid (ABA)