EMBL, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China.
Fish Shellfish Immunol. 2011 Jul;31(1):29-42. doi: 10.1016/j.fsi.2011.02.016. Epub 2011 Mar 8.
Expressed sequence tags (ESTs) analysis has been shown to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of Portunus trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. A total of 99 unigenes including 64 unigenes (6.00% of 1066 unigenes) in hemocytes library and 35 unigens (6.86% of 510 unigenes) in eyestalk library are identified to be immune genes. These genes are categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. The content and category of immune genes in eyestalk library indicate eyestalk might have unrecognized role in crab immunity. Five immune genes containing multiple protein isoforms are identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). Sequence alignment and phylogenetic analysis reveal PtALF1-7 contain two conserved cysteine residues and might be encoded by multiple genomic loci. PtCrustin1-3 share the consensus cysteine motif and are considered as Type I crustins. PtTrx1 possesses the critical structural cysteine residue C⁷³ of Trx-1, while PtTrx2 has the N-terminal mitochondrial translocation signal of Trx-2. Sequence analysis shows PtcSP1-5 contain one clip domain and one partial SP catalytic triad domain. PtKPI1-4 present one typical Kazal domain consisting of six conserved cysteine residues. Some protein isoforms are tissue-specific, which might suggest they have different origins and perform diverse functions. Except PtALF1-3 and PtCrustin1, the other isoformes in this study are firstly identified from P. trituberculatus. Especially, PtTrx2 are firstly identified from crustaceans. Our research will provide useful genomic information of P. trituberculatus and be helpful in understanding the molecular mechanisms of crab immunity.
表达序列标签（EST）分析不仅是发现基因的有效方法，也是基因表达谱性能的有效方法。从三疣梭子蟹的血细胞和眼柄中分别构建了两个全长富集 cDNA 文库，并对其进行随机测序，以收集基因组信息并鉴定参与免疫防御反应的基因。总共鉴定出 99 个基因，其中血细胞文库中有 64 个基因（1066 个基因中的 6.00%），眼柄文库中有 35 个基因（510 个基因中的 6.86%）被鉴定为免疫基因。这些基因分为六大类，即抗菌肽、氧化还原蛋白、黑化相关蛋白、伴侣蛋白、凝结蛋白和其他免疫因子。眼柄文库中免疫基因的含量和类别表明，眼柄在螃蟹免疫中可能具有未被认识的作用。鉴定并表征了 5 个含有多个蛋白同工型的免疫基因，包括抗脂多糖因子（PtALF1-7）、甲壳质（PtCrustin1-3）、硫氧还蛋白（PtTrx1-2）、 clip 结构域丝氨酸蛋白酶（PtcSP1-5）和 kazal 型蛋白酶抑制剂（PtKPI1-4）。序列比对和系统发育分析表明，PtALF1-7 含有两个保守的半胱氨酸残基，可能由多个基因组位点编码。PtCrustin1-3 共享保守的半胱氨酸基序，被认为是 I 型甲壳质。PtTrx1 具有 Trx-1 的关键结构半胱氨酸残基 C⁷³，而 PtTrx2 具有 Trx-2 的线粒体转运信号的 N 端。序列分析表明，PtcSP1-5 含有一个 clip 结构域和一个部分 SP 催化三联体结构域。PtKPI1-4 呈现出由六个保守半胱氨酸残基组成的典型 Kazal 结构域。一些蛋白同工型具有组织特异性，这可能表明它们具有不同的起源和执行不同的功能。除了 PtALF1-3 和 PtCrustin1，本研究中的其他同工型是首次从三疣梭子蟹中鉴定出来的。特别是，PtTrx2 是首次从甲壳类动物中鉴定出来的。我们的研究将为三疣梭子蟹提供有用的基因组信息，并有助于理解螃蟹免疫的分子机制。
