原壳小球藻番茄红素ε环化酶基因的克隆和分析

Cloning and analysis of the gene encoding lycopene epsilon cyclase in Chlorella protothecoides CS-41

摘要:

摘要：【目的】番茄红素ε环化酶（lycopene epsilon cyclase）是叶黄素代谢途径中处于分支位点的关键酶。它催化线性的番茄红素选择性环化，形成胡萝卜素，再进一步合成叶黄素（lutein）。本研究的目标是克隆原壳小球藻（Chlorella protothecoides CS-41）LCYE基因的全长cDNA，通过该基因的生物学信息分析预测其表达产物的空间结构与功能位点，并验证该表达产物的生物学活性。【方法】采用cDNA末端快速扩增（rapid-amplification of cDNA ends）和RT-PCR技术克隆原壳小球藻LCYE基因的全长cDNA序列。分别采用PredictProtein、Pfam HMMs和Swiss-Model等在线分析软件分析LCYE的基本生物学信息，预测蛋白质功能位点和空间结构。利用pET28-a(+)构建LCYE基因的原核表达质粒pET-LCYE，并转入大肠杆菌BL21（DE3），再利用IPTG诱导LCYE基因超量表达。此外，携带pAC-LYC质粒的大肠杆菌工程菌能够积累番茄红素，可用于验证LCYE基因编码蛋白的酶活。【结果】获得了原壳小球藻的LCYE基因的cDNA序列，长2107 bp，GenBank登录号为FJ752528。序列分析表明：它含有1731 bp的完整开放阅读框（ORF），编码576个氨基酸，与高等植物和其他藻类的LCYE有很高的相似性，其中相似性最高的是莱茵衣藻（Chlamydomonas reinhardtii XM001696477.1），达到67%。第48～459个氨基酸残基为一个典型的番茄红素环化酶蛋白（Lycopene cyclase protein）结构域（pfam05834），第261～284个氨基酸残基为一个典型的环化酶保守的模体。SDS-PAGE检测表明，LCYE基因在大肠杆菌BL21（DE3）中得到了超量表达。携带pAC-LYC质粒的大肠杆菌工程菌在获得LCYE基因后，其颜色从粉红色变为黄色。【结论】原壳小球藻LCYE基因的全长cDNA为2107 bp，预测出番茄红素ε环化酶所特有的保守功能区域，构建了它的三维结构模型。同时，阐明了小球藻和衣藻的亲缘关系。最后，证实了本研究克隆到的LCYE基因编码的蛋白具有番茄红素ε环化酶的功能与活性。

Abstract:

Abstract: [Objective] Lycopene epsilon cyclase (LCYE) is the key enzyme in the lutein synthesis pathway and catalyses linear lycopene to form cyclic ε-carotene, a presucursor of lutein. We aimed to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41, to predict the functional sites and the three-dimensional structure of LCYE through bioinformatics analysis and to confirm its activities and functions. [Method] We used RACE (rapid-amplification of cDNA ends) essay and RT-PCR for the cloning of the full-length cDNA of LCYE from C. protothecoides CS-41. The online software such as PredictProtein, Pfam HMMs and Swiss-Model were used in bioinformatics analysis of the amino acid sequence of LCYE protein. We constructed the expression vector for LCYE gene with pET-28a(+) and transformed into Escherichia coli BL21(DE3). Furthermore, the E. coli strain containing the pAC-LYC plasmid which could accumulate lycopene was used for the functional confirmation of LCYE from C. protothecoides CS-41. [Results] A 2107 bp cDNA（GenBank Accession No.FJ752528）sequence was cloned with 1731 bp open reading frame, encoding a putative LCYE, from C. protothecoides CS-41. Homology studies showed that the deduced amino acid sequence of LCYE gene had a significant similarity with the corresponding sequences of other green algae and higher plants. It shared the highest sequence identity, up to 67%, with the LCYE gene from Chlamydomonas reinhardtii. One typical lycopene cyclase protein domain (Pfam05834) was predicted between the 48th -459th amino acid. In addition, the sequence between 261th -284th was one typical conserved lycopene cyclase protein motif. The SDS-PAGE result showed that the LCYE gene was over-expressed in Escherichia coli BL21(DE3) after the addition of IPTG. The prokaryotically expressed LCYE protein was able to transfer the color of the E. coli strain containing the pAC-LYC plasmid from pink to yellow. [Conclusion] The full-length cDNA sequence of LCYE gene was successfully cloned with the size of 2107 bp. Several typical motifs were found and the three-dimensional structure of LCYE was constructed from Bioinformatics analysis. The generated phylogenetic tree showed the closest relationship between C. protothecoides CS-41 and C. reinhardtii among the listed organisms. Finally, the expression product of the LCYE gene cloned in the study was confirmed to hold the function and activity of lycopene epsilon cyclase.