日本结缕草ZjHCT基因的克隆及转录自激活检测

Cloning and Autonomous Transcriptional Activation Testing of ZjHCT Gene from Zoysia japonica

莽草酸/奎宁酸羟基肉桂酰转移酶(shikimate/quinate hydroxycinnamoyl transferase,HCT)是木质素生物合成的关键酶之一,也是不同类型木质素相互转化的调控物质。日本结缕草(Zoysia japonica)是一种应用广泛的暖季型坪草。以日本结缕草为材料,利用PCR技术克隆日本结缕草ZjHCT基因完整编码区,结果显示,日本结缕草ZjHCT基因编码区全长1 284 bp,共编码428个氨基酸。生物信息学分析显示,ZjHCT蛋白组件中,随机线圈和α螺旋含量最多,其不具备信号肽,定位在细胞质内的概率最大。利用DNA重组技术,构建pBGKT7-ZjHCT载体并进行Y2H Gold酵母菌株转化及自激活检测,证明ZjHCT在Y2H酵母菌株中不具备自激活功能,可在日本结缕草中进行互作蛋白筛选实验,为深入研究日本结缕草木质素合成及提高日本结缕草草坪草耐践踏性,改善坪用质量提供帮助。

Shikimate/quinate hydroxycinnamoyl transferase(HCT)is one of the key enzymes in lignin biosynthesis and the regulator of the interconversion between different types of lignin.Zoysia japonica is a widely-used warm-season turfgrass.Taking Zoysia japonica as material,the complete coding region of ZjHCT gene was cloned from Zoysia japonica by PCR technology.The results showed that the whole length of coding region of ZjHCT gene was 1 284 bp in Zoysia japonica,which encoded 428 amino acids.Bioinformatics analysis showed that the random coil andα-helix were the most abundant protein among all ZjHCT protein components,which didn't have signal peptide and was most likely to be located in the cytoplasm.The pBGKT7-ZjHCT vector was constructed and Y2HGold yeast strain was transformed and detected the self-activation based on DNA recombinant technology,indicating that ZjHCT protein had no self-activation activity in Y2H yeast strains.Interacting protein screening experiments could be carried out in Zoysia japonica,which might provide help for further studying the lignin synthesis of Zoysia japonica and improving trampling tolerance and turf quality.