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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基础之上,采用RACE技术,克隆获得油茶脂酰辅酶A脱氢酶基因的全长c DNA序列,命名为Co ACAD(基因登录号KJ910338)。该基因c DNA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 k D,理论等电点p I为8.47,具有2个比较明显的跨膜区和酪氨酸蛋白激酶活性位点LVHGDFRIDNLVF,存在5个亚结构域;在Co ACAD基因c DNA全长序列的基础上构建表达载体,其中原核表达载体在宿主细胞BL21(DE3)中成功诱导表达,获得表观分子量约为93 k D的目的蛋白;实时荧光定量PCR分析表明,Co ACAD基因在果实膨大期和成熟期上调表达,预示着Co ACAD基因可能在种子发育过程中参与能量供应过程的调控。
Based on the constructed transcriptome and expression profile database, the full-length c DNA of Camellia oleifera (Aspergillus oleifera) seeds was cloned by using RACE technique. Sequence, named Co ACAD (Gene Accession Number KJ910338). The full-length cDNA of this gene was 2702 bp in length and contained a 2487 bp open reading frame encoding 828 amino acids with a molecular weight of 92.4113 kD and a theoretical isoelectric point pI of 8.47 with two distinct transmembrane regions Based on the full-length cDNA sequence of Co ACAD gene, an expression vector was constructed. The prokaryotic expression vector was successfully induced in the host cell BL21 (DE3) to obtain the expression vector The results showed that the Co ACAD gene was up-regulated during the enlargement and maturation of fruit, indicating that Co ACAD gene may be involved in the energy supply process during seed development.