天麻抗真菌蛋白GAFP-Ⅰ的一级结构和cDNA克隆

Primary Structure and cDNA Cloning of the Antifungal Protein GAFP-I from Gastrodia elata

天麻抗真菌蛋白ＧＡＦＰ－Ⅰ是从天麻（ＧａｓｔｒｏｄｉａｅｌａｔａＢｌ）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（Ａｒｍｉｌａｒｉａｍｅｌｅａ）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６００ｂｐ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４２９ｂｐ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ｍａｎ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４％一致。ＧＡＦＰ－Ⅰ由１２９个氨基酸组成，富含Ａｓｎ（１９）、Ｇｌｙ（１４），２９和５２位间有一对双硫键，计算分子量为１３９５８Ｄａ。ＧＡＦＰ－Ⅰ与雪花莲（Ｇａｌａｎ?

The Gastrodia Antifungal Protein GAFP-I was purified from topical corm of Tian Ma( Gastrodia 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 corm was amplified to be a specific cDNA of about 600 bp through a 3′- RACE experiment with the degenerated primer. The cDNA was directly cloned into pGEM-T vectors and sequenced. The nucleotide sequenceof cDNA and C-terminal amino acid sequence to be -ASG of GAFP-I determined with carboxypeptidase A indicate that the cDNA contains a 429 bp region encoding GAFP-I of 129 aa and a C-terminal extenting peptide of 14 aa. Futhermore, peptides were separated and purified from hydroxylamine cleavaged and α- chymotrypsin digested GAFP-I, and sequenced by automa tic Edman degradation. The determined aa sequence was found to be 98 4% identical to the one deduced from nucleotide sequence of the cDNA. GAFP-I is a single polypeptide of 129 aa, rich in Asn(19)and Gly(14), with a disulfide bridge between the 29th and 52th aa and a calculated Wt of 13958 Da. A high aa sequence similarity was found between GAFP-I and the snowdrop( Galanthus nivalis L.)lectin LECGNA 2, the match of aa was 65%, including an identically located disulfide bridge. The nucleotide sequence of cDNA encoding GAFP-I has been submitted to GenBank database under an accession number AF123309.