Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide
Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide
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拟青蟹血细胞脂多糖差异表达基因的鉴定
DOI：
10.1016/j.fsi.2009.10.017
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发表时间：
2010-01-01
影响因子：
4.7
通讯作者：
Wang, Ke-Jian
中科院分区：
文献类型：
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作者：
Chen, Fang-Yi;Liu, Hai-Peng;Wang, Ke-Jian
虽然蟹Scylla paramamosain在中国已经培养了很长时间，但螃蟹对细菌感染的反应却知之甚少。为了分离S. paramamosain对细菌脂多糖（LPS）响应的差异表达基因，我们构建了正向抑制减法杂交（SSH） cDNA文库，并对上调基因进行了鉴定。对SSH库中721个中等规模的克隆进行了测序。其中，271个潜在功能基因通过NCBI BLAST检索得到识别，并利用AmiGO对Gene Ontology数据库进行分类，根据不同的生物学过程将其分为7组。在271个基因中，269个可翻译的DNA序列被预测为蛋白质，并使用CD-Search服务和BLASTp搜索推定的氨基酸序列以寻找保守结构域和蛋白质。271个基因中，179个（66.1%）被标注参与不同的生物过程，92个（33.9%）被归类为功能未知的基因群。结果表明，271个基因中仅有18个（6.6%）在其他甲壳类动物中报道过，且大部分基因与BLASTn结果的相似性较低，表明253个基因为首次在S. paramamosain中发现。从SSH文库中筛选两个上调基因，分别克隆sp -超氧化物歧化酶（Sp-Cu-ZnSOD）基因和Sp-serpin基因，进行全长cDNA序列克隆和体内表达研究。采用实时荧光定量PCR技术分析两个基因在LPS胁迫过程中的差异表达模式。我们发现，与对照组相比，这两个基因在lps挑战的螃蟹中显著表达。综上所述，本研究主要提供了S. paramamosain在LPS作用下与不同生物学过程相关的上调基因的数据，未来将研究可能参与S. paramamosain先天免疫防御的有趣基因或蛋白质。2009爱思唯尔有限公司版权所有。
Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two upregulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the upregulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future. (C) 2009 Elsevier Ltd. All rights reserved.