多穗柯无色花青素还原酶基因的克隆及其表达与根皮苷含量的相关性分析

Cloning and expression of LAR gene and its correlation with phloridzin content in Lithocarpus polystachyus

目的克隆多穗柯Lithocarpus polystachyus的无色花青素还原酶(leucoanthocyanidin reductase,LAR)基因,并对其表达量与根皮苷含量的关系进行分析。方法根据多穗柯转录组测序的结果(Unigene号:DN30711_c0_g1_i1),利用PCR法扩增获得多穗柯LAR基因的全长cDNA序列,进行生物信息学分析,采用实时荧光定量PCR(qRT-PCR)法检测LAR基因的表达量,使用UPLC法测定根皮苷的含量,应用SPSS 18.0软件分析LAR基因表达量与根皮苷含量间的相关关系。结果多穗柯LAR基因的cDNA全长1053 bp,包含一个完整的开放阅读框,编码350个氨基酸。该蛋白不存在跨膜区域,定位于细胞质中。多穗柯LAR蛋白属于PCBER_SDR_a家族,与栓皮栎LAR蛋白具有较高的相似性(95%),且亲缘关系最近。多穗柯的根皮苷含量与LAR基因的表达量呈正相关(P<0.05)关系。结论首次克隆得到多穗柯LAR基因,并证实多穗柯根皮苷含量与LAR基因的表达量呈正相关,为揭示多穗柯中根皮苷的生物合成机制奠定理论和技术基础。

Objective To clone the leucoanthocyanidin reductase(LAR)gene of Lithocarpus polystachyus,and analyze the relationship between LAR gene expression level and phloridzin content.Methods According to the results of L.polystachyus transcriptome sequencing(unigene:DN30711_c0_g1_i1),the full-length cDNA sequence of LAR gene was amplified by PCR and the bioinformatics analysis was carried out.Its expression was detected by quantitative Real-time PCR(qRT-PCR).The phloridzin content of L.polystachyus was measured by UPLC method and the correlation between LAR gene expression and phloridzin content was analyzed by SPSS 18.0 software.Results The full-length cDNA of the LAR gene was 1053 bp and contained a complete open reading frame that encoded 350 amino acids.This protein did not exist a transmembrane domain and was localized in the cytoplasm.The LAR protein of L.polystachyus was the number of PCBER_SDR_a family and had a high similarity(95%)to the LAR protein of Quercus suber and their genetic relationship was close.The phloridzin content of L.polystachyus was positively correlated with the expression of LAR gene(P<0.05).Conclusion The LAR gene of L.polystachyus was cloned for the first time.It was confirmed that the content of phloridzin was positively correlated with the expression of LAR gene of L.polystachyus,which laid a theoretical and technical basis for revealing the biosynthesis mechanism of phloridzin of L.polystachyus.