板栗脂质转运蛋白基因的克隆及表达  

Cloning and Expression of Chestnut Lipid Transfer Protein

作　　者：唐征[1] 杨凯[2] 冯永庆[1] 曹庆芹[2] 沈元月[1] 秦岭[1] 

机构地区：[1]北京农学院植物科学技术学院,北京102206 [2]北京农学院生物技术系,北京102206

出　　处：《林业科学》2010年第4期43-48,共6页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(30872039)

摘　　要：利用RACE技术从板栗短雄花序突变体中扩增得到660bp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-gamiTM2(DE3)中,IPTG诱导5h大量表达约为30ku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。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 ThioFusionTM 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-gamiTM 2 （ 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.

关 键 词：板栗 脂质转运蛋白 实时定量RT-PCR 抑菌功能 

分 类 号：S718.46[农业科学—林学] Q943.2[生物学—植物学]