湘油15 PGIP9蛋白基因的克隆和蛋白序列结构分析及原核表达  

Cloning,Sequence and Structure Analysis of Polygalacturonase Inhibiting Protein Gene(PGIP 9) of Brassica napus Xiangyou 15 and Prokaryotic Expression

作　　者：刘睿洋[1] 皇甫海燕 靳芙蓉 官春云 

机构地区：[1]湖南农业大学油料作物研究所,长沙410128 [2]国家油料作物改良中心湖南分中心,湖南长沙410128

出　　处：《作物研究》2012年第2期134-138,共5页Crop Research

基　　金：国家"863"基金项目(2006AA10A112);农业部"948"基金项目(2011-G23)

摘　　要：运用生物信息学软件对湘油15 PGIP9基因的核苷酸、蛋白氨基酸序列进行分析,并对其蛋白结构进行预测。结果表明:湘油15 PGIP9编码区CDS长1 011 bp,编码336个氨基酸的开放阅读框,分子量为37.5kDa,等电点为7.9。N端1～22个氨基酸是信号肽,且这一区域疏水性较强,具有5个潜在的N-糖基化位点。N端和C端还各具有4个参与二硫键形成的半胱氨酸残基。二级结构显示有11个α-螺旋,14个β-延伸和25个无规则卷曲。中心LRR结构域由6个串联的LRR基序组成。随后将PGIP9的CDS序列亚克隆到原核表达载体pET-32a(+)中,构建pET-32a-PGIP9重组表达质粒,并转化E.coil BL21(DE3),25℃,终浓度为0.2 mmol/L和0.5 mmol/L的IPTG诱导2 h,都成功的表达了融合蛋白pET-32a-PGIP9,其分子量约为52kDa,发现主要以包涵体形式存在,没有可溶形式的蛋白表达。Ribonucleotide and amino acid sequence of Xiangyou 15 PGIP were analyzed,the protein structure were predicted by biological information software.CDS of Xiangyou 15 PGIP 9 code area was 1 011 bp,which encoded 336 amino acid of open reading frame with a molecular mass of 37.5 kDa and isoelectric point of 7.9.1~22 amino acids were predicted to the N-terminal signal peptide with stronger hydrophobic region.The deduced amino acids sequence contained 5 potential N-glycosylation sites and 4 cysteine residues on N-terminal and C-terminal respectively,which involved in constituting the disulfide linkages.Secondary structure displayed it contained 11 α-helices,14 β-extension and 25 random coils.The center LRR structural domain was composed of 6 tandem LRRs motifs.Later,CDS of PGIP 9 was subencoded into the prokaryotic expression vector pET-32a（＋）constructing recombinant expression plasmid pET-32a-PGIP9 and was transformed into Escherichia coli BL21（DE3）.The fusion protein pET-32a-PGIP9 was successfully expressed under final concentration 0.2 mmol/L and 0.5 mmol/L IPTG at 25℃ inducing for 2 h.The molecular weight of fusion protein was 52 kDa,it mainly appeared as inclusion bodies,not appeared as soluble protein.

关 键 词：甘蓝型油菜 多聚半乳糖醛酸酶抑制蛋白 原核表达 序列分析 结构预测 

分 类 号：Q78[生物学—分子生物学]