巨龙竹肉桂醇脱氢酶基因(DsCAD)的克隆及功能分析

Cloning and Functional Analysis of Cinnamyl Alcohol Dehydrogenase Gene from Dendrocalamus sinicus

肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)是木质素生物合成途径的关键酶。研究根据巨龙竹转录组数据设计的一对特异引物,运用逆转录PCR(RT-PCR)技术从巨龙竹茎秆中克隆得到肉桂醇脱氢酶基因DsCAD,该基因包含1080 bp的完整c DNA开放阅读框,编码359个氨基酸,理论相对分子质量38689.6,等电点6.18。利用生物信息学方法对DsCAD进行分析,结果显示:DsCAD氨基酸序列包含CAD超家族的保守结构域,具有植物CAD基因的典型特征;其与禾本科植物CAD基因的同源性较高,亲缘关系较近,其中与孝顺竹的同源性高达95%。荧光定量PCR检测结果显示DsCAD基因在巨龙竹竹笋中的表达量要高于茎秆,而在茎秆中的表达量有明显高于叶。

Cinnamyl alcohol dehydrogenase （CAD） is one of the key enzymes in lignin biosynthesis.In this study, a CAD gene （designated as DsCAD） was obtained from the culm of Dendrocalamus sinicus by RT-PCR with a pair of special primers designed based on the transcriptome date.The DsCAD gene has a 1080bp open reading frame （ORF） that is predicted to encode a protein of 359 amino acids with predicted molecular mass of 38689.6 and a basic isoelectric point of 6.18.DsCAD gene was analyzed by bioinformatics tools.The results showed that the DsCAD encoding sequence has the conserved domain of CAD superfamily and it has typical characteristics of CAD genes in plants.Dendrocalamus sinicus has more closer relative relationship with Poaceae plants and the DsCAD sequence shared high sequence identity（95%） with Bambusa multiplex.Real time PCR analysis showed that the DsCAD was most strongly expressed in the shoots.This result will provide useful information for lignins biosynthesis research of Dendrocalamus sinicus in future.