Chan S M
Department of Zoology, The University of Hong Kong, China.
FEBS Lett. 1998 Oct 9;436(3):395-400. doi: 10.1016/s0014-5793(98)01148-x.
Degenerate primers were derived from the amino acid sequence in the DNA binding domain of the Drosophila ecdysone receptor (DmEcR). Several partial cDNAs were amplified from the shrimp epidermis by reverse transcription polymerase chain reaction (RT-PCR). One of these fragments shows the highest amino acid sequence homology to the insect ecdysone inducible gene E75. This partial cDNA was used as a probe to screen the swimming leg cDNA library of the shrimp, Metapenaeus ensis. A 3.6 kb cDNA clone was obtained. The longest open reading frame of this cDNA consists of 606 amino acids and its deduced amino acid sequence has all five domains typical of a nuclear receptor. The putative polyadenylation signal is located at about 400 bp 3' to the stop signal. The deduced amino acid sequence of this cDNA shows the highest identity to that of the E75A reported in Manduca sexta, Galleria melonella, Drosophila melanogaster, and Choristoneura fumiferana. Based on the amino acid sequence comparison, the shrimp nuclear receptor is considered the insect homologue of E75A. Northern blot analysis shows that the shrimp E75 is expressed in the epidermis, eyestalk and the nerve cord of the pre-molt shrimp. Moreover, E75 transcripts can be detected in the epidermal tissues of early pre-molt shrimp by in situ hybridization. To determine whether the shrimp could also express other E75s like the insects, 5' end RACE and RT-PCR were performed on epidermal cDNA of a single shrimp. Subcloning and DNA sequence determination of the PCR products confirmed the presence of two other forms of E75 (tentatively called E75C and E75D) in shrimp. By RT-PCR, different levels of E75 expression can be detected in the epidermis, nerve cord and the eyestalk of early pre-molt shrimp. In addition to the different levels of expression of the shrimp E75s in the epidermis, the pattern of their expression is also different during the molting cycle. This is the first report on the cloning of a shrimp nuclear receptor superfamily member.
简并引物源自果蝇蜕皮激素受体（DmEcR）DNA结合结构域中的氨基酸序列。通过逆转录聚合酶链反应（RT-PCR）从虾表皮中扩增出几个部分cDNA。其中一个片段与昆虫蜕皮激素诱导基因E75的氨基酸序列同源性最高。这个部分cDNA被用作探针来筛选虾（新对虾）游泳足的cDNA文库。获得了一个3.6 kb的cDNA克隆。该cDNA最长的开放阅读框由606个氨基酸组成，其推导的氨基酸序列具有核受体典型的所有五个结构域。推测的聚腺苷酸化信号位于终止信号下游约400 bp处。该cDNA推导的氨基酸序列与烟草天蛾、大蜡螟、黑腹果蝇和云杉卷叶蛾中报道的E75A的氨基酸序列一致性最高。基于氨基酸序列比较，该虾核受体被认为是E75A的昆虫同源物。Northern印迹分析表明，虾E75在蜕皮前虾的表皮、眼柄和神经索中表达。此外，通过原位杂交可在蜕皮前早期虾的表皮组织中检测到E75转录本。为了确定虾是否也能像昆虫一样表达其他E75，对一只虾的表皮cDNA进行了5'端RACE和RT-PCR。PCR产物的亚克隆和DNA序列测定证实了虾中还存在另外两种形式的E75（暂称为E75C和E75D）。通过RT-PCR，在蜕皮前早期虾的表皮、神经索和眼柄中可检测到不同水平的E75表达。除了虾E75在表皮中的表达水平不同外，它们在蜕皮周期中的表达模式也不同。这是关于虾核受体超家族成员克隆的首次报道。
