基于同源克隆技术分离玉米漆酶基因片段

漆酶是木质素生物合成途径的关键酶,其高度保守的铜离子结合区以及N末端糖基化位点使利用同源克隆技术成为可能。本研究利用GeneBank中公布的黑麦草漆酶基因全长cDNA序列及在玉米数据库中的比对结果设计特异引物,以8份AS系列玉米自交系为模板进行PCR筛选。经过提取质粒、测序及对推导的氨基酸序列进行比对,发现所克隆基因片段中含有保守的H-A-H基元和N末端糖基化位点,为以后玉米漆酶基因全长的克隆以及饲用玉米改良打下了基础。

Isolation of Laccase Fragments from Maize by Homology-based Cloning

Laccase is one of key enzymes involved in lignin biosynthesis pathway. Homology-based cloning method is available with the highly conserved copper-binding sites for each of the ligand residues and conserved potential N-linked glycosylation sites in laccases. In the present study, we retrieved laccase cDNA sequences from Lolium perenne and maize from the databases. Subsequently, the gene-specific primers to the maize sequences were designed and used for PCR amplification with eight AS set maize inbreds. Conserved H-A-H motifs and potential N-linked glycosylation sites were found in the cloned fragments, which provided the basis for both cloning full-length laccases genes of maize and improving the quality of forage maize.