Watanabe T, Kono M
Laboratory of Molecular Biology of Marine Organisms, University of Tokyo, Japan.
Zoolog Sci. 1997 Feb;14(1):65-8. doi: 10.2108/zsj.14.65.
To identify and characterize a chitinase related to molting in the Kurumia prawn Penasus japonicus, we searched for chitinase-encoding cDNAs expressed in cuticular tissues. Using two degenerate oligonucleotide primers derived from the two conserved regions of the chitinase protein family, a RT (reverse transcription)-PCR product was obtained. This product was used as a probe to screen a cDNA library from a mixture of the tall fan and blade-two tissues which consist mainly of chitinous exoskeleton and underlying epidermis. A positive cDNA clone was analyzed for the sequence. This clone contains an open reading frame for a protein (named Pjchi-2) of 527 amino acids which exhibits sequence similarity to known chitinases. A typical signal sequence could not be found in the Pjchi-2 sequence. Significant accumulation of Pjchi-2 mRNA was detected in the mixture of the tall fan and blade prior to molting, whereas the transcript level was much lower during the intermolt stage. This observation suggests that Pjchi-2 plays a role in molting. The mRNA was not detected in the hepatopancreas. This expression pattern of Pjchi-2 makes a contrast to that of Pjchi-1 which encodes another chitinase family protein in P. japonicus, and is expressed in the hepatopancreas but not in the tall fan or blade.
为了鉴定和表征与日本对虾蜕皮相关的几丁质酶，我们在表皮组织中搜索了编码几丁质酶的cDNA。使用从几丁质酶蛋白家族的两个保守区域衍生的两个简并寡核苷酸引物，获得了一个RT（逆转录）-PCR产物。该产物用作探针，从主要由几丁质外骨骼和下层表皮组成的高扇叶和叶片两种组织的混合物中筛选cDNA文库。对一个阳性cDNA克隆进行了序列分析。该克隆包含一个527个氨基酸的蛋白质（命名为Pjchi-2）的开放阅读框，该蛋白质与已知的几丁质酶具有序列相似性。在Pjchi-2序列中未发现典型的信号序列。在蜕皮前的高扇叶和叶片混合物中检测到Pjchi-2 mRNA的显著积累，而在蜕皮间期转录水平要低得多。这一观察结果表明Pjchi-2在蜕皮中起作用。在肝胰腺中未检测到该mRNA。Pjchi-2的这种表达模式与Pjchi-1形成对比，Pjchi-1编码日本对虾中的另一种几丁质酶家族蛋白，在肝胰腺中表达，但在高扇叶或叶片中不表达。
