Branchini Bruce R, Southworth Tara L, DeAngelis Jennifer P, Roda Aldo, Michelini Elisa
Department of Chemistry, Connecticut College, New London, Connecticut 06320, USA.
Comp Biochem Physiol B Biochem Mol Biol. 2006 Oct;145(2):159-67. doi: 10.1016/j.cbpb.2006.06.001. Epub 2006 Jun 8.
The cDNA encoding the luciferase from the Italian firefly Luciola italica was cloned using reverse transcriptase-PCR and a gene-specific primer set based on the DNA sequence of Luciola mingrelica. The cDNA sequence of L. italica luciferase was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Phylogenetic analysis of the protein sequence demonstrated that this luciferase is closely related to that of other fireflies of the Lampyridae family, particularly within the Luciolinae subfamily, showing 96% homology to luciferases from the fireflies Hotaria unmunsana and Hotaria parvula. The specific activity of the L. italica luciferase was 78% of the North American enzyme, after correction for emission color differences. The bioluminescence emission of the Italian firefly is pH sensitive with maxima at 566 nm and 614 nm at pH 7.8 and 6.0, respectively. Interestingly, the total bioluminescence output was approximately 2-fold greater than that of P. pyralis luciferase due to differences in turnover characteristics evidenced by extended light emission decay kinetics. We expect that this newly discovered luciferase will be suitable for a wide range of bioluminescence applications including in vivo imaging and multiplex assays.
利用逆转录聚合酶链反应（RT-PCR）以及基于明纹扁萤（Luciola mingrelica）DNA序列设计的基因特异性引物对，克隆了编码意大利萤火虫（Luciola italica）荧光素酶的cDNA。意大利萤火虫荧光素酶的cDNA序列长度为1647个碱基对，开放阅读框为548个氨基酸。对该蛋白质序列进行的系统发育分析表明，这种荧光素酶与萤科其他萤火虫的荧光素酶密切相关，尤其是在熠萤亚科内，与芒萤（Hotaria unmunsana）和小萤（Hotaria parvula）的荧光素酶具有96%的同源性。在对发射颜色差异进行校正后，意大利萤火虫荧光素酶的比活性为北美荧光素酶的78%。意大利萤火虫的生物发光发射对pH敏感，在pH 7.8和6.0时，最大发射波长分别为566 nm和614 nm。有趣的是，由于延长的发光衰减动力学所证明的周转特性差异，意大利萤火虫的总生物发光输出比萤火虫（Photinus pyralis）荧光素酶大约高2倍。我们预计，这种新发现的荧光素酶将适用于包括体内成像和多重检测在内的广泛生物发光应用。
