Cloning and Characterization of a cDNA Encoding an Antimicrobial Protein from Mung Bean Seeds

綠豆種子抗微生物蛋白質之cDNA選殖與特性分析

陳冠仲(Kuan-Chung Chen) ； 林景堉(Ching-Yu Lin) ； 鍾美珠(Mei-Chu Chung) ； 官振群(Cheng-Chun Kuan) ； 宋賢一(Hsien-Yi Sung) ； 鄒篪生(Sanson C.S.Tsou) ； 郭忠吉(C.George Kuo) ； 陳慶三(Ching-San Chen)

《Botanical Bulletin of Academia Sinica》 43卷4期 (2002/10) Pp. 251-259

https://doi.org/10.7016/BBAS.200210.0251

摘要

我們自一對近似等基因品系的綠豆種子表現差異cDANs中，分离出一種抗微生物蛋白質的cDNA，命名為VrCRP(GenBank accession no. AF326687)。成熟的VrCRP蛋白質屬于鹼性、富含半光胺酸的小分子量蛋白質，分子量約為5.9kDa，等電點在8.23。蛋白質一級結構中一共有八個半光胺酸，可組成四對雙硫鍵。在生物活性分析方面，發現VrCRP蛋白質對大腸桿菌有毒性，并于低濃度(3.42μM)下即可破壞昆虫細胞，VrCRP蛋白質也抑制真菌（立枯絲核菌）的生長。免疫組織定位及原位雜交發現VrCRP主要集中在綠豆種子的種皮之中，尤其是儲積在薄壁細胞層(parenchyma cell)。基于VrCRP的生物活性以及蛋白質位於綠豆種皮，我們推測VrCRP在綠豆種子中可扮演病源入侵時的一重要防禦角色。

關鍵字

抗微生物蛋白質 ； 免疫組織定位 ； 原位雜交 ； 綠豆

並列摘要

A cDNA clone, VrCRP (GenBank accession no. AF326687), encoding an antimicrobial protein was isolated from the differentially expressed cDNAs between a pair of near isogenic lines of mid-matured mung bean seeds. Northern blot analysis showed that VrCRP gene is preferentially expressed in bruchid-resistant developing mung bean seeds. The mature VrCRP is a basic cysteine-rich protein with a predicted molecular mass of 5.9 kDa and a calculated isoelectric point of 8.23. Eight cysteine amino acids present in the primary structure of VrCRP compose four disulfide bridges. Biological analysis showed that VrCRP is toxic to E. coli and completely arrested growth of insect cell (Spodopterafrugiperda, Sf21) at a low concentration of 3.42 μM. VrCRP also inhibited the growth of Rhizoctonia solani. Immunolocalization and in situ hybridization indicated that VrCRP was predominantly present in the parenchyma cells of the seed coat. Based on the biological activities and the protein localization, it is suggested that VrCRP may act as an evolutionary barrier in mung bean seeds that is important in coping with invasions of pathogens and herbivores.

並列關鍵字

Antimicrobial protein ； Immunolocalization ； In situ hybridization ； Mung bean