油茶亲环素基因的鉴定与其在大肠杆菌中的表达

Identification of a gene for cyclophilin of Camellia oleifera and its expression in Escherichia coli

利用构建的油茶近成熟种子cDNA文库,分离鉴定了一条油茶亲环素基因的cDNA序列;该序列全长975bp(base pair),含有一个621bp的开放码框,编码207个氨基酸,将其命名为co-cyp(GenBank登录号:FJ377540)。该基因与其它物种的亲环素基因具有较高的同源性,结构分析表明该蛋白的二级结构以片层为主,并伴有α-螺旋、β转角和大量的无规则卷曲;其N端有一个强疏水性的信号肽序列,具有跨膜结构和较好的柔性;还具有N-连接糖基化位点、蛋白激酶C磷酸化位点、酪氨酸激酶Ⅱ磷酸化位点、酪氨酸激酶磷酸化位点、N-连接肉豆蔻酰位点、脯氨酸顺反异构酶标志序列、环孢菌素(CsA)结合位点(Trp)以及植物亲环素基因特有的7个氨基酸特征序列KSGKPLH。该蛋白属于亲环素B族,为球状分泌蛋白。在获得油茶亲环素基因全长cDNA的基础上,构建了该基因的原核表达重组子pET30b-CyP,在大肠杆菌BL21(DE3)中诱导表达,获得了约21kDa的油茶亲环素蛋白。本研究对揭示油茶种子的油脂合成和抗逆性能等生物学规律具有重要意义。

Cyclophilins are folding enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases（PPIases） that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins.The full-length cyclophilin gene clone was isolated,cloned,identified,and expressed using the cDNA library conducted by the nearly matured Camellia oleifera seeds.Its size was 975 bp containing an open reading frame（ORF） of 621 bp,encoded a 22.24-kDa protein of 207 amino acid residues,and was named co-cyp.The secondary structure of the protein mainly contains extended strands acompanying with alpha helixs,beta turns,and numerous random coils.It contains a 26-amino acid singal sequence in the N-terminal domain,and has a transmembrane region and fine flexibility.It also possesses N-glycosylation sites,protein-kinase C phosphorylation sites,Casein kinaseII phosphorylation sites,Tyrosine kinase phosphorylation site,N-myristoylation sites,Cyclophilin-type peptidyl-prolyl cis-trans isomerase signature YKGSKFHRIIPSFMLQGG,seven amino acid residues KSGKPLH of plant cyclophilins,et al.It showed that the protein belonged to CyPB,and was a globular secretory protein.It shares overall identities of 80%~94% with those of some higher plants.On the basis of the full-length cDNA,the approximately 21 kDa protein was obtained via the constructed recombinant pET30-CyP in E.Coli strain BL21（DE3） induced by IPTG under the optimum expressed conditions.This study may be of very importance for the pathway of lipid biosynthesis and stress response of Camellia oleifera.