Molecular Cloning of a Chitinase Gene from the Ovotestis of Kuroda’s Sea Hare Aplysia kurodai
Molecular Cloning of a Chitinase Gene from the Ovotestis of Kuroda’s Sea Hare Aplysia kurodai
复制标题
DOI：
10.4236/abb.2016.71005
复制
发表时间：
2016-01
期刊：
影响因子：
--
通讯作者：
Gaku Matsunaga;Syuuji Karasuda;Ryo Nishino;H. Fukushima;M. Matsumiya
中科院分区：
文献类型：
--
作者：
Gaku Matsunaga;Syuuji Karasuda;Ryo Nishino;H. Fukushima;M. Matsumiya
在这项研究中，我们成功地从黑田海兔的卵睾丸中克隆并测序了一个几丁质酶基因。利用逆转录聚合酶链反应（RT-PCR）和cDNA末端5′和3′快速扩增系统，从黑田鼠卵睾丸中获得了一个1352 bp的几丁质酶基因（AkChi）。AkChi包含一个1263 bp的开放阅读框，编码421个氨基酸。根据氨基酸序列预测的结构域结构为一个n端信号肽和一个糖苷水解酶（GH）家族18几丁质酶的催化结构域。AkChi的氨基酸序列与加利福尼亚海兔（applysia californica）的酸性哺乳动物几丁质酶的氨基酸序列的比较分析显示，同源性最高，为83%。经胰蛋白酶酶切得到的卵睾丸几丁质酶有119个残基与推导出的AkChi氨基酸序列一致。我们用RT-PCR方法检测了AkChi在黑田鼠各组织中的表达情况，发现AkChi仅在卵睾丸中表达。利用AkChi和已知GH家族18几丁质酶的氨基酸序列进行系统发育树分析，发现AkChi与具有几丁质结合结构域的软体动物几丁质酶分离。据我们所知，这是首次从海兔中克隆卵睾丸几丁质酶的研究。
In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.