Molecular Population Genetics Group, Temasek Life Sciences Laboratory, National University of Singapore, 117604 Republic of Singapore.
BMC Genomics. 2010 Jun 4;11:356. doi: 10.1186/1471-2164-11-356.
Fish diseases caused by pathogens are limiting their production and trade, affecting the economy generated by aquaculture. Innate immunity system is the first line of host defense in opposing pathogenic organisms or any other foreign material. For identification of immune-related genes in Asian seabass Lates calcarifer, an important marine foodfish species, we injected bacterial lipopolysaccharide (LPS), a commonly used elicitor of innate immune responses to eight individuals at the age of 35 days post-hatch and applied the suppression subtractive hybridization (SSH) technique to selectively amplify spleen cDNA of differentially expressed genes.
Sequencing and bioinformatic analysis of 3351 ESTs from two SSH libraries yielded 1692 unique transcripts. Of which, 618 transcripts were unknown/novel genes and the remaining 1074 were similar to 743 known genes and 105 unannotated mRNA sequences available in public databases. A total of 161 transcripts were classified to the category "response to stimulus" and 115 to "immune system process". We identified 25 significantly up-regulated genes (including 2 unknown transcripts) and 4 down-regulated genes associated with immune-related processes upon challenge with LPS. Quantitative real-time PCR confirmed the differential expression of these genes after LPS challenge.
The present study identified 1692 unique transcripts upon LPS challenge for the first time in Asian seabass by using SSH, sequencing and bioinformatic analysis. Some of the identified transcripts are vertebrate homologues and others are hitherto unreported putative defence proteins. The obtained immune-related genes may allow for a better understanding of immunity in Asian seabass, carrying out detailed functional analysis of these genes and developing strategies for efficient immune protection against infections in Asian seabass.
由病原体引起的鱼类疾病限制了其生产和贸易，影响了水产养殖所产生的经济。先天免疫系统是宿主抵御致病生物或任何其他外来物质的第一道防线。为了鉴定亚洲海鲈（Lates calcarifer）的免疫相关基因，这是一种重要的海洋食用鱼类，我们在孵化后 35 天对 8 条个体注射细菌脂多糖（LPS），这是一种常用的先天免疫反应诱导剂，并应用抑制性消减杂交（SSH）技术选择性扩增脾脏 cDNA 的差异表达基因。
从两个 SSH 文库的 3351 个 EST 序列测序和生物信息学分析中，得到了 1692 个独特的转录本。其中，618 个转录本为未知/新基因，其余 1074 个与 743 个已知基因和 105 个公共数据库中未注释的 mRNA 序列相似。共有 161 个转录本被归类为“对刺激的反应”类别，115 个转录本被归类为“免疫系统过程”。我们鉴定了 25 个与 LPS 刺激相关的免疫相关过程的显著上调基因（包括 2 个未知转录本）和 4 个下调基因。定量实时 PCR 证实了 LPS 刺激后这些基因的差异表达。
本研究首次通过 SSH、测序和生物信息学分析，在亚洲海鲈中鉴定出 LPS 刺激后的 1692 个独特转录本。其中一些鉴定的转录本是脊椎动物同源物，另一些是迄今未报道的推定防御蛋白。获得的免疫相关基因可以帮助更好地了解亚洲海鲈的免疫，对这些基因进行详细的功能分析，并制定有效的免疫保护策略，以防止亚洲海鲈感染。
