花生光敏色素 AhphyA 和AhphyB 基因的克隆及原核表达研究

摘要

【目的】克隆了花生（Arachis hypogaea L．）的2个光敏色素基因，并对其进行生物信息学分析，构建这2个基因的原核表达载体，在大肠杆菌中诱导表达蛋白，为进一步纯化蛋白制备抗体以及研究这2个基因在花生发育中的作用奠定基础。【方法】首先，根据花生转录组中编码光敏色素 A 基因与光敏色素 B 基因的部分序列，以花生叶片中提取的总 RNA 为模板，通过 RT-PCR 与 RACE 技术对这2个基因的全长 ORF 序列进行克隆；然后，使用 MEGA软件分析花生光敏色素与其他物种中光敏色素的亲缘关系；构建 AhphyA、AhphyB 的原核表达载体，转化大肠杆菌 BL21并诱导蛋白表达。【结果】2个基因全长 ORF 分别为3378 bp 和3456 bp，分别编码1125和1151个氨基酸的蛋白；其氨基酸序列与大豆的光敏色素 A 蛋白和光敏色素 B 蛋白的相似性分别为88％和90％；经 SDS-PAGE 检测，在大肠杆菌中 2个基因均能受 IPTG 诱导表达产生蛋白。【结论】利用 RT-PCR 与 RACE技术从花生中克隆得到了 2个光敏色素基因，成功构建了原核表达载体，并使其在大肠杆菌中得到了高效表达。%[Objective]Two peanut phytochrome genes were cloned and analyzed.Prokaryotic ex-pression within fusion proteins in E .coli was carried out,which could be helpful for future study on antibody preparation and functional analysis of these genes during peanut pod development.[Method]Two partial sequences of phytochrome genes were obtained from peanut transcriptome sequences.RT-PCR,3′and 5′RACE were used to acquire the full length ORFs of these two genes.Phylogenetic relationships between two peanut phytochrome and phytochromes from other plants were analyzed using MEGA.The expression vectors were constructed and IPTG was used to induce the fusion proteins expression in E .coli BL21.[Results]The full length of AhPhyA and AhPhyB ORF was 3 378 bp and 3 456 bp,encoding proteins with 1 125 and 1 151 amino acid, respectively.The similarity of AhPhyA and AhPhyB to GmPhyA and GmPhyB was 88% and 90%,respectively.The SDS-PAGE result showed that fusion proteins were successfully induced in E .coli by IPTG.[Conclusion]The full length ORF of AhPhyA and AhPhyB were obtained. The expression vectors were constructed using partial sequences of AhphyA and AhphyB .The fusion proteins were successfully induced in E .coli BL21 by IPTG.