三疣梭子蟹NF-κB家族基因Relish和Dorsal的克隆及表达特征

为研究Relish和Dorsal在三疣梭子蟹免疫过程中所起到的作用, 研究利用RACE技术克隆获得三疣梭子蟹Relish(Pt-Rel)、Dorsal基因(Pt-Dor) cDNA全长, 并通过实时荧光定量PCR技术分析了Pt-Rel和Pt-Dor基因在健康蟹不同组织及其原代培养的血淋巴细胞在感染不同微生物后的表达情况。结果显示, Pt-Rel cDNA长3254 bp, ORF长2949 bp, 编码983个氨基酸, Pt-Dor cDNA长2348 bp, ORF长1911 bp, 编码637个氨基酸; 蛋白结构预测分析发现Pt-Rel和Pt-Dor均包含RHD (Rel homology domain)及IPT (Immunoglobulin-like fold, Plexins, TranscriPtion factors)Rel/NF-κB家族蛋白经典结构域。Pt-Rel和Pt-Dor与其他节肢动物Relish、Dorsal氨基酸序列具有很高的相似性; 在系统进化分析中Pt-Rel和Pt-Dor分别与中华绒螯蟹等甲壳动物的Relish、Dorsal聚在一支, 而昆虫类聚在另一支。Pt-Rel和Pt-Dor在检测的6种组织中均有表达, 且2个基因均在血淋巴细胞中表达量最高。蟹血淋巴细胞体外感染实验结果表明, 不同病原微生物对2个基因表达的影响非常相似, 假丝酵母在2h明显诱导了Pt-Rel和Pt-Dor基因的表达, 而金黄色葡萄球菌及溶藻弧菌在感染4h后使Pt-Rel和Pt-Dor基因的表达显著上调。上述研究结果表明Pt-Rel和Pt-Dor基因很有可能参与了三疣梭子蟹的抗感染免疫过程。     

Multiple isoforms of immune-related genes from hemocytes and eyestalk cDNA libraries of swimming crab Portunus trituberculatus

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 C?3 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.     

三疣梭子蟹Profilin基因全长cDNA的克隆与表达分析

三疣梭子蟹（Portunus trituberculatus）是我国沿海重要养殖品种之一,近年来养殖病害呈逐年上升趋势,制约了三疣梭子蟹养殖产业的健康可持续发展。克隆三疣梭子蟹免疫相关基因,研究免疫基因的功能和作用机制,可为三疣梭子蟹养殖病害的防治奠定基础。本研究从三疣梭子蟹血细胞全长cDNA文库中克隆了742 bp 的profilin基因全长cDNA。Profilin全长cDNA中开放阅读框长375 bp,编码125 aa。推导的三疣梭子蟹profilin理论等电点pI 5.87,氨基酸序列与冈比亚按蚊（Anopheles gambiae）profilin同源性最高,序列一致性为42.9%。荧光定量RT-PCR分析结果显示在正常的三疣梭子蟹机体中,血细胞profilin表达水平最高,其次为肝胰脏;在致病菌副溶血弧菌（Vibrio parahaemolyticus）诱导后,血细胞中profilin表达量显著上升（P<0.01）,表明profilin可能参与了三疣梭子蟹的免疫防御反应,是一个免疫相关因子。     

Polymorphisms of anti-lipopolysaccharide factors in the swimming crab Portunus trituberculatus and their association with resistance/susceptibility to Vibrio alginolyticus

Anti-lipopolysaccharide factor (ALF) is an important antimicrobial peptide (AMP) that can bind and neutralize major component of Gram-negative bacteria cell wall, lipopolysaccharide (LPS). Seven isoforms of anti-lipopolysaccharide factors (PtALF1-7) were previously identified from the swimming crab Portunus trituberculatus in our laboratory. Here, polymorphisms of PtALF1-7 were detected and their association with resistance/susceptibility to Vibrio alginolyticus (a main Gram-negative bacteria causing high mortality in P. trituberculatus) were investigated. We identified 127, 96, 103, 53 and 158 single nucleotide polymorphisms (SNPs) in genomic fragments of PtALF1-3, PtALF4, PtALF5, PtALF6 and PtALF7, respectively. Among them, totally sixteen SNPs were significantly associated with resistance/susceptibility to V. alginolyticus (P < 0.05). Of these sixteen SNPs, most were located in introns and noncoding exons, while two synonymous SNPs and one nonsynonymous SNP were in coding exons. Additionally, simple sequence repeats (SSRs) were only identified in introns and noncoding exons of PtALF4, PtALF5 and PtALF7. Although no significant difference of allele frequencies was found, these SSRs had different polymorphic alleles according to the repeat number between susceptible and resistant stocks. After further confirmation, polymorphisms investigated here might be applied as potential molecular markers for future selection of resistant strains to diseases caused by Gram-negative bacteria.     

Cloning and expression pattern analysis of a lipopolysaccharide -and beta-1,3-glucan-binding protein in Portunus trituberculatus

The lipopolysaccharide -and beta-1,3-glucan-binding protein (LGBP) is a pattern recognition receptor, which is fundamental for the innate immune response of crustaceans. A LGBP gene was cloned from the haemocytes of Portunus trituberculatus using SMART RACE methods. The full-length LGBP cDNA (1 378 bp) had a 1 095 bp open reading frame encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138 bp 5' untranslated region (UTR) and a 144 bp untranslated region in the 3' UTR with a 29 bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39,825.24 with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glyco hydro 16 domain was identified. The expression of P. trituberculatus in various tissues were detected through RT-PCR methods. The results showed that the LGBP gene expressed in all the tissues detected, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytes of the group challenged with mixed bacteria were higher than the control group within 48 h. It suggested that the LGBP gene plays an active role in immunologic process against bacterial infection.     

SpALF4: A newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand β-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.     

不同病原菌刺激下三疣梭子蟹主要组织LGBP基因的时序表达

脂多糖-β-1,3-葡聚糖结合蛋白（lipopolysaccharide-β-1,3-glucan binding protein,LGBP）作为一种模式识别受体能够识别革兰氏阴性菌和真菌的细胞壁多糖成分脂多糖和β-1,3-葡聚糖,在甲壳动物的免疫系统中占有重要地位,为了解LGBP在蟹类动物抗菌免疫应答中的作用,采用SYBR Green I实时荧光定量PCR方法,对分别经溶藻弧菌,酵母菌,溶-酵混合菌、金黄色葡萄球菌刺激6 h、12 h、24 h、48 h的三疣梭子蟹血细胞、肝胰腺、肌肉3种组织LGBP基因的相对表达量进行了检测。结果显示,LGBP基因在3种组织中的时序表达量总体趋势基本一致,呈现＂上升-下降-再上升-下降＂的趋势,各实验组在实验6 h时表达量均达到了峰值;不同病原菌刺激下LGBP基因在梭子蟹肝胰腺和血细胞中的表达量明显高于肌肉,且溶-酵混合菌刺激组织LGBP的表达量最高,酵母菌和溶藻弧菌刺激组次之,金黄色葡萄球菌组最低,说明LGBP主要参与蟹类抵抗革兰氏阴性菌和真菌侵入的免疫防御反应,但对革兰氏阳性菌也有一定的作用。     

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.