鬼臼毒素是八角莲的重要药效成分.利用同源克隆和RACE的方法,从八角莲的根中克隆了鬼臼毒素生物合成途径肉桂醇脱氢酶(Cinnamyl alcohol dehydrogenase,CAD)基因,长1098 bp,编码由365个残基组成的氨基酸序列.八角莲CAD蛋白不含有质体定位的信号序列,与毛白杨(Populus tomentosa)、拟南芥(Arabidopsis thaliana)等物种中CAD蛋白的一致性为67.25%;系统发育分析表明,它与拟南芥的AtCAD7、AtCAD8和At-CAD6蛋白亲缘关系较近,聚成一个簇.组织表达分析结果表明,八角莲CAD基因在根和茎中有表达,而叶片中表达不明显.本研究获得的CAD基因,为后续进行详细的功能解析,并进一步应用于鬼臼毒素的遗传调控,奠定了基础.%Objective Podophyllotoxin is an important bioactive substance in D. versipellis.The gene encoding a cinnamyl alcohol dehydrogenase from root of D. versipellis was cloned, and its tissue expression pattern was characterized.Methods A CAD gene was obtained with degenerated primers amplification,3′RACE and 5′RACE from root cDNA,and analyzed by bioinformatic softwares.The expression pattern was predicted through cDNA am-plification from root,stem and leaves. Results A 1098 bp CAD gene encoding a 365 amino acid sequence was cloned.This amino acid sequence contained no plastid targeted signal peptide,had 67.25% similarities with the CAD protein from Populus tomentosa and Arabidopsis thaliana,and could be clustered with AtCAD7,AtCAD8 and AtCAD6 into the same group in phylogenetic analysis.The expression of the CAD gene was detected in root and stem,but not obviously in leaves of D.versipellis.Conclusion CAD is a key enzyme in biosynthesis of podo-phyllotoxin.The CAD gene from D. versipellis was cloned,which lays a foundation for podophyllotoxin genetic engineering.
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