Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase
Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase
复制标题
DOI：
10.1111/j.1751-1097.1999.tb07997.x
复制
发表时间：
1999-08-01
影响因子：
3.3
通讯作者：
Reinach, FC
中科院分区：
文献类型：
--
作者：
Viviani, VR;Silva, ACR;Reinach, FC
在巴西中西部地区，点击甲虫 Pyrearinus termitluminans 的幼虫引发了发光白蚁丘的现象。该幼虫的生物发光 (BL) 光谱（λ(max) = 534 mm）是已知发光鞘翅目中蓝移最严重的光谱之一。我们从幼虫胸灯中分离了 mRNA，并将 cDNA 文库构建到 lambda ZAP II 载体中。切除pBluescript 质粒后获得表达文库。通过光电检测对该文库进行筛选，并分离出一个发出绿色 BL（lambda(max) = 538 nm）的克隆。 2.2 kb cDNA 插入片段包含 543 个残基的开放阅读框，与 Pyrophorus plagiophamus（鞘翅目：Elateridae）的荧光素酶同工酶具有 82% 同源性。正如预期的那样，残基 223 和 247 之间的区域包含用于 BL 颜色测定的推定活性位点，在点击甲虫荧光素酶之间显示出更高程度的同源性，从而引发更接近的 BL 颜色。重组白蚁荧光素酶的体外 BL 光谱也在 538 nm 处达到峰值，并且与天然酶的情况一样，在降低 pH 值时没有显示任何红移。
The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil. The bioluminescence (BL) spectrum of this larva (lambda(max) = 534 mm) is one of the most blue-shifted reported among known luminescent Coleoptera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (lambda(max) = 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.