Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e-5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs. (C) 2009 Elsevier Ltd. All rights reserved.

The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes; (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans. (C) 2009 Elsevier Ltd. All rights reserved.

烟草单半乳糖甘油二酯缺失对茉莉酸生物合成的影响

茉莉酸（JA）是由脂肪酸衍生而来的环戊酮化合物，广泛存在于自然界中，在植物逆境胁迫响应和生长发育调节过程中起重要作用。因此，JA被认为是一种新型植物激素。植物JA生物合成的最初底物是三烯脂肪酸（含有三个双键的十八碳和十六碳脂肪酸，18:3和16:3），这些脂肪酸经过脂氧合酶（LOX）、丙二烯氧化物合酶（AOS）和丙二烯氧化物环化酶（AOC）等一系列酶促反应，最终生成JA。JA生物合成所需要的三烯脂肪酸来自叶绿体膜脂。高等植物叶绿体类囊体膜含有四种极性甘油脂，它们是：单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)、硫代异鼠李糖甘油二酯(SQDG)和磷脂酰甘油(PG)。但是人们尚不清楚JA生物合成所需要的三烯脂肪酸主要来自哪一种膜脂。 最近，我们利用RNA干扰技术获得了烟草MGDG部分缺失的突变体。MGDG是质体中最重要的甘油脂，其含量高达50%，其中含有的三烯脂肪酸约占总脂中三烯脂肪酸含量的65%。本研究的目的是以烟草MGDG缺失的突变体（mgd1）为材料，通过研究MGDG缺失对茉莉酸生物合成的影响，阐明半乳糖脂与JA生物合成的关系。 首先我们对野生型烟草（WT）和mgd1的相关生物学特性进行了研究，包括甘油脂和脂肪酸组成。结果表明，mgd1烟草叶片中MGDG含量降低了57％，同时，其三烯脂肪酸相对含量也大幅度降低。其中十六碳三烯酸（16:3）降低了78%，亚麻酸（18:3）含量减少了28%。因此，由于MGDG缺失，类囊体中的三烯脂肪酸降低了27%。这一结果说明了JA生物合成的底物大幅度减少。 为了说明MGDG缺失导致的三烯脂肪酸含量的减少是否影响到JA的含量，我们利用GC-MS方法比较了WT和mgd1烟草中JA的含量。结果表明，mgd1叶片中的JA含量较WT降低了50%，说明了MGDG的缺失影响了JA的生物合成。 伤害可以诱导JA在短时间内大量合成。我们比较了机械损伤后JA在WT和mgd1叶片中积累的动态过程。伤害同时可以使WT和mgd1叶片中的JA含量增加，并且在1小时达到最大值。但是，JA在两种烟草叶片中增加的幅度不同，WT叶片受伤1小时后JA含量是未受伤时的5倍，而mgd1叶片受伤1小时后，其JA含量只增加了1倍。这些结果说明了MGDG缺失可以严重影响伤害诱导的 JA 的积累，MGDG是JA的生物合成底物的重要来源。 我们进一步研究了MGDG缺失对JA生物合成相关酶基因表达的影响。 LOX1和AOC编码JA生物合成途径中的关键酶LOX和AOC。RT-PCR分析表明mgd1叶片中这两个基因受伤害激活的程度比WT弱。进一步说明突变体中JA合成受到影响。 植物受到伤害时内源JA含量增加，并激活防御基因的表达。我们的结果显示，当植物受伤害后，mgd1叶片中与JA信号转导相关的防御基因HPL，PI-I和PI-II的表达量增加幅度明显低于WT。这说明突变体中JA信号转导途径受到了抑制。 JA在植物对昆虫侵害的防御反应中起重要作用，上述结果表明突变体对伤害响应受到削弱。昆虫饲喂实验显示，棉铃虫更趋向食用mgd1植株叶片，取食mgd1植株的棉铃虫的体重增加较多。这些结果与WT和mgd1在JA含量、防御相关基因表达方面的差异相一致。外源施加茉莉酸甲酯（MeJA）能够恢复mgd1的抗虫性和防御基因的表达，说明JA是恢复mgd1抗虫性所必须的。 上述结果表明MGDG缺失使JA生物合成受到影响，尤其是JA在植物受到伤害后的生物合成。对于这一现象的可能的解释是：MGDG是JA生物合成底物的主要来源，由于mgd1中缺少大量的MGDG，当植物受到伤害时，MGDG不能释放出足够三烯脂肪酸来合成JA，导致其含量降低，破坏了JA信号途径，最终使得植株表现出抗性降低等特性。我们的研究证明了MGDG可以作为JA生物合成的底物来源在JA信号途径中起重要作用。

Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value >= 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.

Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. in this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value <= 5e-3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGF beta 1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XIBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia. (c) 2008 Elsevier Inc. All rights reserved.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.

Avian influenza virus infection induces differential expression of genes in chicken kidney

The pathogenic process of highly pathogenic avian influenza virus (HPAIV) infection is poorly understood. To explore the differential expression of kidney genes as a result of HPAIV infection, two cDNA libraries were constructed from uninfected and infected kidneys by suppression subtractive hybridization (SSH). Fifteen genes including IFN-stimulated genes (ISG12), lymphocyte antigen 6 complex locus E gene (LY6E), matrix Gla protein gene (MGP), lysozyme gene, haemopoiesis related membrane protein I gene, KIAA1259, MGC68696, G6pe-prov protein gene (G6PC), MGC4504, alcohol dehydrogenase gene (ADH), glutathione S-transferase gene (GST), sodium-dependent high-affinity dicarboxylate transporter gene (SDCT), Synaptotagmin XV (SytXV) and two novel genes were found significantly up-regulated or dramatically suppressed. Differential expression of these genes was further identified by Northern blot. Functional analysis indicated that the regulation of their expression might contribute to the pathogenic process of HPAIV infection. In contrast, the increased expression of three IFN-stimulated genes named ISG12, LY6E, and haemopoiesis related membrane protein 1 gene might reflect host defense responses. Further study showed that ISG12 protein failed to directly interact with NS1 protein of HPAIV which expressed simultaneously in the organs where HPAIV replication occurred, by use of BacterioMatch two-hybrid system. Therefore, our findings may provide new insights into understanding the molecular mechanism underlying the pathophysiological process of HPAIV infection in chicken. (c) 2007 Elsevier Ltd. All rights reserved.