Molecular cloning，sequence analysis of phytoene synthase gene from cerasus humilis (bge) sok. and its functional expression in e.coli | 欧李八氢番茄红素合成酶cDNA克隆、序列分析及在大肠杆菌中的功能表达

Chinese. 目的克隆、分析欧李[Cerasus humilis（Bge）Sok]八氢番茄红素合成酶（phytoene synthase，PSY）基因，并利用大肠杆菌异源表达体系验证其功能。方法以欧李果实中分离的总RNA为模板，通过RT-PCR扩增到PSY cDNA中间片段，再利用RACE技术获得该基因cDNA全长并分析其序列；然后利用PCR技术获得欧李PSY cDNA编码区全长，将其克隆到原核表达载体pET-28a(+)，获得重组质粒pET-ChPSY，转化大肠杆菌BL21（DE3）诱导表达，并利用工程菌株验证融合蛋白6×His-PSY的催化活性。结果欧李PSY cDNA全长1 559 bp，最大开放读码框为1 194 bp，可编码398个氨基酸，命名为ChPSY。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70%和65%以上，并且发现欧李PSY的N末端存在1―55个氨基酸残基组成的转运肽信号序列，在37―58和219―240氨基酸区域包含2个跨膜结构域。SDS-PAGE分析表明，原核表达获得了具有较高表达水平的融合蛋白6×His-PSY，相对分子量约为45.8 kD。通过大肠杆菌异源表达证实ChPSY可编码一个功能蛋白，促进工程菌株中β-胡萝卜素含量增加。结论该研究成功地克隆了欧李PSY cDNA全长并在大肠杆菌中功能表达，为进一步研究该酶蛋白特性和欧李果实类胡萝卜素的合成机制奠定了基础。

English. ObjectiveThe present study aimed at cloning and analyzing the phytoene synthase (PSY) gene from Cerasus humilis (Bge) Sok. and verifying its catalytic function by heterologous expression in E. coli containing a β-carotene producing plasmid. MethodUsing the total RNA from the fruit of C. humilis (Bge) Sok. as the template, the cDNA specific fragment of PSY gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and then the full-length cDNA sequence was obtained through rapid amplification of cDNA ends (RACE) techniques and its sequence was analyzed. The full coding sequence of PSY cDNA was amplified using PCR and further subcloned into vector pET-28a (+). The recombinant plasmid pET-ChPSY was expressed in a prokaryotic expression system after its transformation into BL21 (DE3). The catalytic activity of the fusion protein (6×His-PSY) was investigated in E. coli strain engineered to accumulate β-carotene. Result Sequence analysis indicated that the full-length cDNA was 1 559 bp, with an open reading frame of 1 194 bp and encoded a protein of 398 amino acids. The cloned cDNA exhibited a homology of the nucleotides and amino acid sequences of over 70% and 65%, respectively, aligned with PSY genes from other plants. The deduced protein has a signal transit peptide consists of 55 amino acid residues in the N-terminal region and two predicted transmembrane domain in the sites of 37-58 and 219-240 amino acids. The analysis of SDS-PAGE demonstrated that the recombinant fusion proteins (6×His-PSY) was produced at a higher level by prokaryotic expression and migrated at a size of about 45.8 kD. The heterogenous expression in E. coli system confirmed that ChPSY could encode a functional phytoene synthase which could enhance β-carotene content of E. coli engineered to produce β-carotene. Conclusion The PSY gene from Cerasus humilis (Bge) Sok. was successfully cloned and fucntioally expressed in E. coli, which has established a basis for studying the protein characterization and biosynthetic mechanism of carotenoids in fruits of Cerasus humilis (Bge) Sok.