Abstract: The Gastrodia Antifungal Protein GAFP - I was purified from topical corm of Tian Ma ( Gastrodi-a elata), which can strongly inhibit the mycelia growth of seprophytic fungi and plays an important role in the defence mechanism of plants to restrict and prevent the infection of the fungus Armillaria mellea. This paper reports the primary structure and the cDNA nucleotide sequence of GAFP - I. The N - terminal 7 amino acid sequence to be SDRLNSG - was determined by automatic sequencer and used for synthesizing a degenerated primer. The mRNA from the Gastrodia conn was amplified to be a specific cDNA of about 600 bp through a 3' - RACE experiment with the degenerated primer.

Key words: Gastrodia elata, Antifungal protein GAFP-I, cDNA cloning, Hydroxyamine cleavage

摘要： 天麻抗真菌蛋白 Gi-1是从天麻（Gastrodia elat Bl．）顶生球茎分离的 14kD蛋白，它能强烈抑制腐生真菌菌丝的生长，在天麻限制和防止密环菌(Armillaria mellea)侵染球茎的防卫机制中起重要作用。本文报告GAFP-I的一级结构和cDNA克隆的核苷酸序列。首先测得N-末端7个氨基酸序列为SDRLNSG-用以合成一个简并引物，由天麻营养球茎提取RNA，通过 3’-RACE技术扩增到一个600hp的cDNA片段，直接克隆到 pGE-T载体中测定核苷酸序列。根据cDNA核苷酸序列和用羧肽酶A测得GAFP-I的C-末端为-ASG，确认该cDNA含有429 hp编码区，它编码一条129个氨基酸的GAFP-I和14个氨基酸的C-末端扩展肽。同时，分离和纯化GAFP－I经过羟胺裂解和α-糜乳胰蛋白酶消化产生的肽段，经自动Ed－rnan降解程序测出它们的氨基酸序列。结果表明，由此测出的GAFP-I氨基酸序列和由cDNA核苷酸序列推导出的序列98.％一致。GAFP－I由 129个氨基酸组成，富含 Asn（19）、Gy（14），29和52位间有一对双硫键，计算分子量为13958 Da。GAFP－I与雪花莲（Galanthus nivalis L．）凝集素 LECGNA 2的氨基酸序列有很高的相似性，该凝集素 105个氨基酸全序列与GM－I相应序列之间相同和相匹配氨基酸达65％，包括一对定位相同的双硫键。编码GAFP－I的cDNA核苷酸序列已发GenBank数据库：accession number AF123309。

关键词: 天麻, 抗真菌蛋白GAFP-1, 蛋白质一级结构, cDNA克隆, 羟胺裂解