Prapaipong H, Berenbaum M R, Schuler M A
Department of Biochemistry, University of Illinois, Urbana 61801.
Nucleic Acids Res. 1994 Aug 11;22(15):3210-7. doi: 10.1093/nar/22.15.3210.
Detoxification of host plant defensive compounds by larval Lepidoptera is mediated by cytochrome P450 monooxygenases (P450s) such as CYP6B1, which is expressed in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin. Baculovirus-mediated expression of two cloned CYP6B1 cDNAs in lepidopteran cell lines has demonstrated that CYP6B1 isozymes primarily metabolize the linear furanocoumarins, xanthotoxin and bergapten, and not angular furanocoumarins. To characterize the regulatory features of the CYP6B1 transcription unit, we have isolated the first full-length CYP6B1v3 genomic DNA clone from P. polyxenes. The open reading frame of this gene is interrupted by a single intron and is virtually identical to the previously characterized CYP6B1 cDNAs. Primer extension and ribonuclease protection analyses have localized the transcription initiation site to a point 28 nucleotides upstream from the AUG initiation codon. RNase protection analyses on RNA from larvae induced by linear and angular furanocoumarins indicate that transcription of the CYP6B1 gene is induced in insects significantly in response to xanthotoxin and only slightly in response to bergapten. Angular furanocoumarins, such as angelicin, which are not appreciably metabolized by the CYP6B1 gene product, do not significantly induce transcription of this gene. We conclude that this P450 gene is transcriptionally regulated in vivo by at least one of the substrates which the encoded protein metabolizes. Transient expression of CAT fusion constructs in transfected Sf9 lepidopteran cells demonstrates that nucleotides -1 to -838 upstream from the CYP6B1v3 transcription initiation site retain basal and xanthotoxin-inducible transcriptional activities in this heterologous cell line. These data clearly indicate that P. polyxenes has adapted to the presence of furanocoumarins in its host plants by evolving P450 isozymes and regulatory cascades which respond to specific toxins.
鳞翅目幼虫对宿主植物防御性化合物的解毒作用是由细胞色素P450单加氧酶（P450s）介导的，如CYP6B1，它在金凤蝶（黑凤蝶）幼虫中响应黄曲霉毒素（一种线性呋喃香豆素）而表达。杆状病毒介导的两个克隆的CYP6B1 cDNA在鳞翅目细胞系中的表达表明，CYP6B1同工酶主要代谢线性呋喃香豆素、黄曲霉毒素和补骨脂素，而不是角型呋喃香豆素。为了表征CYP6B1转录单元的调控特征，我们从金凤蝶中分离出了第一个全长CYP6B1v3基因组DNA克隆。该基因的开放阅读框被一个内含子打断，并且与先前表征的CYP6B1 cDNA几乎相同。引物延伸和核糖核酸酶保护分析已将转录起始位点定位到AUG起始密码子上游28个核苷酸处的一个点。对由线性和角型呋喃香豆素诱导的幼虫RNA进行的核糖核酸酶保护分析表明，CYP6B1基因的转录在昆虫中对黄曲霉毒素有显著诱导作用，而对补骨脂素只有轻微诱导作用。角型呋喃香豆素，如白芷素，不会被CYP6B1基因产物明显代谢，也不会显著诱导该基因的转录。我们得出结论，这个P450基因在体内受到编码蛋白代谢的至少一种底物的转录调控。CAT融合构建体在转染的Sf9鳞翅目细胞中的瞬时表达表明，CYP6B1v3转录起始位点上游-1至-838的核苷酸在这个异源细胞系中保留了基础和黄曲霉毒素诱导的转录活性。这些数据清楚地表明，金凤蝶通过进化出对特定毒素有反应的P450同工酶和调控级联反应，适应了其宿主植物中呋喃香豆素的存在。
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