‘红肉脐橙’八氢番茄红素合成酶基因的克隆与原核表达

Phytoene Synthase Gene Cloning from Citrus sinensis Osbeck cv. Cara Cara and Its Prokaryotic Expression

【目的】克隆、分析‘红肉脐橙’(Citrus sinensis Osbeckcv.Cara Cara)八氢番茄红素合成酶(phytoenesynthase,PSY)基因,并进行原核表达。【方法】以‘红肉脐橙’果实中分离的mRNA为模板,通过RT-PCR扩增到PSY cDNA片段,并分析其序列;利用PCR技术获得‘红肉脐橙’PSY cDNA的编码区全长,并将其克隆到原核表达载体pET28a(+),获得重组质粒pET-CitPSY转化大肠杆菌BL21(DE3)并诱导表达。【结果】‘红肉脐橙’PSY cDNA长1520bp,最大开放读码框为1308bp,可编码436个氨基酸,核酸序列及其推导的氨基酸序列与已知其它植物PSY核酸、氨基酸序列之间的相似性分别在75%和70%以上,并且发现‘红肉脐橙’PSY的N末端存在转运肽信号序列,成熟的PSY包含1个跨膜结构域。原核表达获得了具有较高表达水平的融合蛋白6×His-PSY,Western-blot试验证实表达的融合蛋白能与抗6×His的单抗发生特异性反应,分子量约为52kD。【结论】该研究为进一步开展柑橘果实色泽分子改良奠定了基础。

[Objective]The present study aimed at cloning and analyzing the PSY gene of Citrus sinensis Osbeck cv. Cara Cara and expressing it in the prokaryote. [Method] Using the mRNA from the fruit of Cara Cara as the template, the eDNA ofPSY gene was amplified by Reverse Transcription Polymerse Chain Reaction （RT-PCR） and its sequence was analyzed. The full coding sequence of PSY eDNA was cloned using PCR and further subeloned into pET28a （＋）. The recombinant plasmid pET-CitPSY was expressed in a prokaryotic expression system after its transformation into BL21 （DE3）. [Result] Sequence analysis indicated that the eDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in plants up to more than 75% and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The fusion protein （6 × His-PSY） was produced at a high level by prokaryotic expression. The results of Western-blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The fusion protein molecular mass was about 52 kD. [ Conclusion ] This study has established a basis for molecular improvement of Citrus fruit color.