摘 要 ：本研究以国审油茶(Camellia oleifera)良种‘华硕’种子为材料，在已构建的转录组和表达谱数据库的基础之上，采用RACE技术，克隆到油茶脂酰辅酶A脱氢酶基因的全长cDNA序列，命名为CoACAD (基因登录号KJ910338)。该基因cDNA全长为2702 bp，含有2487 bp 的开放读码框，编码828个氨基酸，分子量为92.4113 kDa，理论等电点pI为8.47，具有两个比较明显的跨膜区和酪氨酸蛋白激酶活性位点“LVHGDFRIDNLVF”，存在五个亚结构域；在基因cDNA全长序列的基础上，成功地构建了表达载体，其中原核表达载体在宿主细胞BL21(DE3)上成功诱导表达，获得表观分子量约为93 kDa相应目的蛋白；实时荧光定量PCR分析表明，CoACAD基因在果实膨大期和成熟期上调表达，预示着CoACAD基因可能参与种子发育过程能量供应过程的调控。
Abstract:In this paper, using state trial oil-tea variety (Camellia oleifera ‘Huashuo’) as material, a full-length cDNA of acyl-CoA dehydrogenase genes in seeds was cloned using RACE technique based on the transcriptome and expression profiling database of C. oleifera seeds. It was named as CoACAD (GenBank accession number KJ910338). The cDNA of CoACAD gene was found to be 2702 bp with an open reading frame of 2487 bp encoding 828 amino acid residues. The molecular mass of the CoACAD protein was 92.4113 kDa with theoretical pI of 8.47. CoACAD protein has two obvious transmembrane domains and contained a typical tyrosine protein kinase (PTK) active site, containing protein kinase domain, aminoglycoside phosphotransferase (APH) domain, ACAD_N domain, ACAD_C domain and ACAD Center domain. The expression vector of CoACAD were constructed successfully, and the BL21 (DE3) bacteria harboring pET30a-CoACAD was induced to express the recombinant protein which was about 93 kDa. The relative expression abundance of CoACAD at 13 different developmental stages of C.oleifera seeds was analyzed using real-time quantitative PCR (qPCR). The CoACAD gene are both up-regulated expression in the C.oleifera seeds’ enlargemental and mature period, which may play be involved in regulation of energy supply at different developmental stages of C. oleifera seeds.