结球甘蓝雌蕊调控转录因子SPT基因的克隆与序列分析

Moclecular Cloning and Sequence Analysis of SPT Gene Transcription Factor Regulation of Pistil from Cabbage

以结球甘蓝E1为材料,提取花蕾总RNA,反转录cDNA。根据拟南芥SPT基因设计引物,采用同源克隆的方法从中克隆SPT基因序列1 085 bp,开放阅读框1 062 bp。通过cDNA推导得到的氨基酸序列分析表明,BoSPT编码353个氨基酸残基,预测分子量为37.67 kD,pI为6.83。经过EcoRⅠ和KpnⅠ限制酶双酶切后,构建原核表达质粒pET43.1a-BoSPT转化表达菌株E.coli Rosetta(DE3),通过SDS-PAGE检测该蛋白的表达。经Smart-embl预测其具有bHLH家族结构域,位于序列第173~221位氨基酸残基处。进化树表明结球甘蓝BoSPT与拟南芥AtSPT和筷子芥AlSPT的亲缘关系较近。BoSPT基因的原核表达得到纯化的融合蛋白。

Taking cabbage(Brassica oleracea L.)E1 as materials,We extracted total RNA from capullo,reverse transcribed cDNA. According to SPT gene sequence of Arabidopsis,primers were designed and 1 085 bp SPT gene with 1 062 bp open reading frame(ORF)was cloned by ho-mology cloning techniques. The deduced BoSPT protein contained 353 amino acids,with a molecu-lar weight of 37.67 kD and pI of 6.83. After double enzyme of EcoRI and KpnI restriction enzymes, and then construct the recombinant plasmids pET43.1a-BoSPT. After transformation to E. coli Ro-setta(DE3),the expression of recombinant proteins were detected via SDS-PAGE. The structural analysis of BoSPT though Smart-embl showed that it contained bHLH family domain,which located at the position of 173-221 amino acid residues,The phylogenetic tree indicated that the BoSPT had close genetic relationship with AtSPT and AlSPT. Prokaryotic expression showed that the molecular mass of BoSPT protein was purified.