Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase.
Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase.
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发表时间：
2002
期刊：
影响因子：
3.3
通讯作者：
M. Lorenzen;Susan J. Brown;R. Denell;R. Beeman
中科院分区：
文献类型：
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作者：
M. Lorenzen;Susan J. Brown;R. Denell;R. Beeman
利用眼色突变体及其相应的基因作为可评分的标记系统，促进了果蝇等昆虫转化技术的发展。在红粉甲虫中，目前唯一可用的生殖系转化系统是利用外源标记基因EGFP，由眼睛特异的启动子驱动。为了利用眼部色素沉着标记的优势，我们决定在突变体拯救的基础上开发一种针对Tribolium的转化子选择系统。对果蝇眼色基因朱砂(色氨酸加氧酶)和朱砂(犬尿氨酸3-单加氧酶)的赤霉菌同源基因进行了克隆和鉴定。Tcv和Tccn的概念翻译分别与它们各自的果蝇同源基因有71%和51%的同源性。我们利用RNA干扰(RNAi)技术表明，缺乏Tcv或Tccn基因产物的卡氏锥虫幼虫也缺乏野生型幼虫中观察到的有色眼斑。通过重组作图，测试了5个可用的眼睛颜色突变与Tcv或Tccn的连锁。未发现候选突变与Tccn相关。然而，在TCV和白眼突变白色之间发现了紧密的联系，在这里被重新命名为朱砂(白色)(v(W))。分子分析表明，v(W)甲虫的TCV编码区有80%缺失。这些观察结果表明，Tribolium的眼睛只由全色蛋白着色，而不是由蕨类化合物着色，并表明TCV作为生殖系转换标记具有潜在的实用价值。
The use of eye-color mutants and their corresponding genes as scorable marker systems has facilitated the development of transformation technology in Drosophila and other insects. In the red flour beetle, Tribolium castaneum, the only currently available system for germline transformation employs the exogenous marker gene, EGFP, driven by an eye-specific promoter. To exploit the advantages offered by eye-pigmentation markers, we decided to develop a transformant selection system for Tribolium on the basis of mutant rescue. The Tribolium orthologs of the Drosophila eye-color genes vermilion (tryptophan oxygenase) and cinnabar (kynurenine 3-monooxygenase) were cloned and characterized. Conceptual translations of Tc vermilion (Tcv) and Tc cinnabar (Tccn) are 71 and 51% identical to their respective Drosophila orthologs. We used RNA interference (RNAi) to show that T. castaneum larvae lacking functional Tcv or Tccn gene products also lack the pigmented eyespots observed in wild-type larvae. Five available eye-color mutations were tested for linkage to Tcv or Tccn via recombinational mapping. No linkage was found between candidate mutations and Tccn. However, tight linkage was found between Tcv and the white-eye mutation white, here renamed vermilion(white) (v(w)). Molecular analysis indicates that 80% of the Tcv coding region is deleted in v(w) beetles. These observations suggest that the Tribolium eye is pigmented only by ommochromes, not pteridines, and indicate that Tcv is potentially useful as a germline transformation marker.