Department of Applied Biology, College of Agriculture and Life Science, Agriculture and Life Sciences Research Institute, Kangwon National University, Chuncheon, 200-701, Korea.
Mol Biol Rep. 2012 May;39(5):5151-9. doi: 10.1007/s11033-011-1311-3. Epub 2011 Dec 13.
To isolate antimicrobial-related genes from the beet armyworm, Spodoptera exigua, we performed GeneFishing, a polymerase chain reaction (PCR)-based differential display technique. An attacin-like complementary DNA (cDNA) including a 3'-untranslated region was identified from among 18 over-expressed genes in microbial-infected larvae. The full-length attacin cDNA from S. exigua cDNA (Seattacin) was cloned using rapid amplification of cDNA ends PCR. The attacin-like cDNA transcript was 765 nucleotides in length, and the predicted polypeptide was 254 amino acids in length with a calculated molecular mass of 27.6 kDa and an isoelectric point of 6.44. The protein sequence of the attacin-like cDNA showed high identity to that of Trichoplusia ni (61.2%). The amino acid sequence identity of Seattacin to the orthologous proteins in Bombyx mori, Manduca sexta, Heliothis virescens, Hlicoverpa armigera, Hyphantria cunea, Hyalophora cecropia, and Drosophila melanogaster was 61.2, 46.1, 44.5, 42.2, 39.5, 45.1, and 24.0%, respectively. To examine possible immune functions of the attacin-like cDNA, its expression was investigated by reverse transcriptase PCR analysis after challenging S. exigua with microorganisms. The attacin-like cDNA was expressed at high levels 12 h post-infection, and its expression was slightly induced 4-8 h post-infection compared to control larvae inoculated with sterile water. Furthermore, induced Seattacin showed biological activity against several bacteria including Escherichia coli DH5α, Pseudomonas cichorii, Bacillus subtilis, and Listeria monocytogenes. These results suggest that the attacin-like cDNA of S. exigua codes for antimicrobial peptides.
为了从甜菜夜蛾（Spodoptera exigua）中分离出与抗菌相关的基因，我们采用基因钓鱼（GeneFishing）技术，即一种基于聚合酶链反应（PCR）的差异显示技术。从微生物感染幼虫中过表达的 18 个基因中鉴定出一个包含 3'-非翻译区的抗菌肽类似 cDNA。使用快速扩增 cDNA 末端 PCR 从 S. exigua cDNA 中克隆全长抗菌肽 cDNA（Seattacin）。抗菌肽类似 cDNA 转录本长 765 个核苷酸，预测的多肽长 254 个氨基酸，计算分子量为 27.6 kDa，等电点为 6.44。抗菌肽类似 cDNA 的蛋白质序列与斜纹夜蛾（Trichoplusia ni）的高度相似（61.2%）。Seattacin 与家蚕（Bombyx mori）、烟青虫（Manduca sexta）、棉铃虫（Heliothis virescens）、棉铃实夜蛾（Hlicoverpa armigera）、舞毒蛾（Hyphantria cunea）、大绿蝇（Hyalophora cecropia）和黑腹果蝇（Drosophila melanogaster）的同源蛋白的氨基酸序列同源性分别为 61.2%、46.1%、44.5%、42.2%、39.5%、45.1%和 24.0%。为了研究抗菌肽类似 cDNA 的可能免疫功能，通过反转录 PCR 分析检测其在微生物刺激后的表达情况。抗菌肽类似 cDNA 在感染后 12 小时高水平表达，与接种无菌水的对照幼虫相比，感染后 4-8 小时略有诱导。此外，诱导的 Seattacin 对包括大肠杆菌 DH5α、菊欧文氏菌（Pseudomonas cichorii）、枯草芽孢杆菌（Bacillus subtilis）和李斯特菌（Listeria monocytogenes）在内的几种细菌具有生物活性。这些结果表明，S. exigua 的抗菌肽类似 cDNA 编码抗菌肽。
