作 者 ：唐征 杨凯 冯永庆 曹庆芹 沈元月 秦岭

期 刊 ：林业科学 2010年 46卷 4期 页码：43-48

关键词：板栗；脂质转运蛋白；实时定量RT-PCR；抑菌功能；

Keywords:Castanea mollissima, lipid transfer protein (LTP), real-time quantitative RT-PCR, resistance function,

摘 要 ：

利用 RACE 技术从板栗短雄花序突变体中扩增得到 660 bp 的板栗脂质转运蛋白（lipid transfer protein，LTP）cDNA 片段。该 cDNA 编码 118 个氨基酸，具有 8 个位置保守的半胱氨酸（C）残基及 26 个氨基酸的信号肽，它与棉花、草莓脂质转运蛋白氨基酸序列相似性为 63%，基因序列已提交数据库（GenBank），其登录号分别为 FJ490676（基因）和 ACL01093（蛋白）。荧光定量分析表明板栗短雄花序突变体较正常雄花序的脂质转运蛋白表达量更高。将板栗脂质转运蛋白基因插入到硫氧还蛋白融合表达载体 pET32a(+)中，构建了板栗的原核表达载体 pET-LTP，在大肠杆菌菌株 Rosetta-gami^TM2（DE3）中，IPTG 诱导 5 h 大量表达约为 30 ku 的融合蛋白，通过 Ni²⁺-chelating sepharose fast flow 柱纯化的融合蛋白具有抑制板栗镰刀菌属病原真菌孢子萌发的功能。

Abstract:

By using rapid amplification of cDNA end ( RACE ) technigue, a cDNA was isolated from the mutation with short staminate inflorescence of Castanea mollissima and was designated as lipid transfer protein (LTP) gene. This cDNA, 660 bp, encoded a 118-amino acid polypeptide with 8 cysteine residues conserved, and a 26-amino acid signal peptide. The deduced polypeptide sequence has submitted to GenBank, the accession No. of gene and protein sequence are AY574218 and AAS79106, respectively. It has 63% similarity to the LTP in cotton and strawberry. Quantitative fluorescence analysis showed the expression level of LTP in short male inflorescence of chestnut was higher than that in normal sample. The gene encoding chestnut LTP was inserted into the ThioFusion^TM expression vector pET32a (+) and constructed the prokaryotic expression vector of pET-LTP. After induced by IPTG for 5 hours, an approximately 30 ku fusion protein was expressed abundantly in the host strain of Rosetta-gami^TM2(DE3). The recombinant LTP protein purified with Ni²⁺-chelating Sepharose Fast Flow column, revealed its microbial inhibition effect on the spore germination of chestnut pathogen Fusarium.