Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors, which show homology with the Drosophila Toll protein and play key roles in detecting various non-self substances and then initiating and activating immune system. In this report, the full length of the first bivalve TLR (named as CfToll-1) is presented. CfToll-1 was originally identified as an EST (expressed sequence tag) fragment from a cDNA library of Zhikong scallop (Chlamys farreri). Its complete sequence was obtained by the construction of Genome Walker library and 5' RACE (rapid amplification of cDNA end) techniques. The full length cDNA of CfToll-1 consisted of 4308 nucleotides with a polyA tail, encoding a putative protein of 1198 amino acids with a 5' UTR (untranslated region) of 211 bp and a 3'UTR of 500 bp. The predicted amino acid sequence comprised an extracellular domain with a potential signal peptide, nineteen leucine-rich repeats (LRR), two LRR-C-terminal (LRRCT) motifs, and a LRR-N-terminal (LRRNT), followed by a transmembrane segment of 20 amino acids, and a cytoplasmic region of 138 amino acids containing the Toll/IL-1R domain (TIR). The deduced amino acid sequence of CfToll-1 was homologous to Drosophila melanogaster Tolls (DmTolls) with 23-35% similarity in the full length amino acids sequence and 30-54% in the TIR domain. Phylogenetic analysis of CfToll-1 with other known TLRs revealed that CfToll-1 was closely related to DmTolls. An analysis of the tissue-specific expression of the CfToll-1 gene by Real-time PCR showed that the transcripts were constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill. The temporal expressions of CfToll-1 in the mixed primary cultured haemocytes were observed after the haemocytes were treated with 1 mu g ml(-1) and 100 ng ml(-1) lipopolysaccharide (LPS), respectively. The expression of CfToll-1 was up-regulated and increased about 2-fold at 6 h with the treatment of 1 mu g ml(-1) LPS. The expression of CfToll-1 was down-regulated with the treatment of 100 ng ml(-1) LPS. The results indicated that the expression of CfToll-1 could be regulated by LPS, and this regulation was dose-dependent. (c) 2006 Elsevier Ltd. All rights reserved.

A toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interteukin-IR (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5 h post-Vibrio anguillorum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum. (c) 2008 Elsevier Ltd. All rights reserved.

中国明对虾Toll样受体基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。自1993 年对虾白斑病暴发以来，中国明对虾的养殖一直一蹶不振。引起对虾大规模死亡的原因是多方面的，其主要原因是养殖环境恶化、对虾种质退化和抗病力下降。因此，深入开展对虾免疫机制研究，并在此基础上寻找对虾疾病防治的有效方法，改良种质和培育抗病品系，已成为对虾养殖业走可持续发展之路的当务之急。 Toll 样受体（Toll-like receptors, TLRs）家族是进化保守的哺乳动物模式识别蛋白（pattern recognition receptors, PRR），在先天免疫系统中起着非常重要的作用。本研究采用同源克隆和RACE（rapid amplification of cDNA ends）技术从中国明对虾中克隆到Toll 样受体同源基因，并将其命名为FcToll。它全长4115 bp，3’UTR 包含16 个poly A 尾巴，开放阅读框编码931 个氨基酸的多肽。预测的该多肽包含典型的Toll 样受体结构，分为胞外区、跨膜区和胞内区。其中胞外区有信号肽，有16 个富含亮氨酸的重复序列eucine-rich repeats, LRR），并含有2个LRR-C 末端基序和2 个LRR-N 末端基序；跨膜区是23 个氨基酸的一次跨膜结构域；胞内区是含有139 个氨基酸的TIR 结构域（Toll/Interleukin-1R）。克隆 发现FcToll 的基因组结构包含5 个外显子和4 个内含子。系统发生分析揭示FcToll归属于“昆虫型”的无脊椎动物Toll 样受体家族。组织分布研究发现FcToll 在中国明对虾中是组成型表达的，在淋巴器官中表达量较显著。分别利用不同病原体刺激健康的中国明对虾，Real-time PCR 发现该基因在刺激后表达水平呈现不同的表达谱：灭活鳗弧菌（Vibrio anguillarum）注射后5 小时，该基因表达显著 上调；而WSSV（white spot syndrome virus）注射后该基因表达则迅速下调，感染后23 小时内其表达水平均低于对应时间点的对照组。这就表明FcToll 可能参与中国明对虾的先天免疫防御，尤其可能参与入侵弧菌的免疫应答。

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus

Double-stranded RNA-activated protein kinase (PKR) plays an important rote in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In the present study, a PKR-like gene was cloned and characterized from rare minnow Gobiocypris rarus. The full length of the rare minnow PKR-like (GrPKZ) cDNA is 1946 bp in Length and encodes a polypeptide of 503 amino acids with an estimated molecular mass of 57,355 Da and a predicted isoelectric point of 5.83. Analysis of the deduced amino acid sequence indicated that the mature peptide contains two Zalpha domains and one S_TKc domain, and is most similar to the crucian carp (Carassius auratus) PKR-like amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed that GrPKZ mRNA expression is at low levels in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus, GrPKZ expression was up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). Following infection with Aeromonas hydrophila, GrPKZ transcripts were induced at 24 h post-injection (P < 0.05) and returned to control levels at 120 h post-injection. These data imply that GrPKZ is involved in antiviral defense and Toll-like receptor 4 signaling pathway in bacterial infection. (C) 2008 Elsevier Ltd. All rights reserved.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

非洲爪蟾早期胚胎发育中PEDF和PEDF-R的功能以及vestigial-like家族表达图式的研究

PEDF 蛋白（Pigment epithelium-derived factor）又名“色素上皮源因子”或 “色素上皮衍生因子”，为一个多功能性分泌糖蛋白，前人研究表明PEDF 蛋白 具有神经保护性、免疫调节、抑制新生血管生成以及抑制肿瘤恶化等多种功能。 PEDF-R 是PEDF 的受体， 属于PNPLA2 （ Patatin-like phopholipase domain-containing 2 family）蛋白家族的一个新成员，PEDF 蛋白与其结合后会激 活PEDF-R 的磷脂酶A2 活性。本研究中，我们描述了非洲爪蟾PEDF 和PEDF-R 基因的表达图式及其在胚胎发育中的可能功能。RT-PCR 结果显示PEDF 是非母 源性表达，而PEDF-R 则是母源性表达的。原位杂交实验表明它们均在神经系统 中特异表达，但PEDF-R 的表达区域更加广泛，在鳃弓、眼泡和耳泡中也有表达。 通过mRNA 过表达和Morpholino(MO)阻断蛋白合成等手段发现，PEDF 功能获 得和功能缺失后胚胎几乎不受影响。然而PEDF-R 过表达后胚胎向注射一侧弯 曲，TUNEL 凋亡检测实验发现这些胚胎在注射一侧发生了凋亡。这两个基因神 经表达的特异性表明它们可能在早期神经发育中有重要功能。TUNEL 结果暗示 着PEDF-R 可能是一个与凋亡信号通路相关的受体。PEDF 功能获得和缺失并未 导致胚胎明显的表型，这表明PEDF 在非洲爪蟾中可能还存在其他的受体来行使 与PEDF-R 不同功能的途径。 果蝇的vestigial 基因编码一个转录辅助因子，在果蝇中只有一个成员，即 vestigial(vg)基因。在脊椎动物中有四个vestigial 同源基因，即vestigial-like 1,2,3,4_(vgl-1,2,3,4)。Vestigial 蛋白能作为辅助因子与果蝇中的Scalloped(Sd)蛋白 或者哺乳动物中的TEF 蛋白结合成复合体，通过Sd/TEF 蛋白的TEA/ATTS 结构 域与DNA 结合，从而调节下游基因的转录。本研究中，我们克隆了非洲爪蟾 vestigial-like 家族的四个成员，并对其在爪蟾胚胎发育过程中的表达进行研究。 RT-PCR 显示vgl-2 和vgl-3 是合子型表达的，vgl-1、vgl-4 则是母源性表达。原位 杂交显示：vgl-1 主要在神经管背部、耳泡和眼泡中表达；vgl-2 则是在肌肉、第 一二鳃弓、脊索中特异表达；vgl-3 神经胚时期在后脑有强的表达信号，从神经 胚后期到尾芽期后脑部位的表达几乎消失了，而在胚胎的头部以及神经管中开始 有微弱的表达；vgl-4 的表达较广泛，在神经管、眼泡、耳泡、肌肉以及脊索中 均有表达。在爪蟾中这四个成员的表达图式各不相同，提示它们有可能与其行使 组织特异性基因调控的功能相关，上述结果将有助于对vestigial-like 家族基因在 胚胎发育中的功能研究。

非洲爪蟾早期胚胎发育中PEDF和PEDF-R的功能以及vestigial-like家族表达图式的研究

PEDF 蛋白（Pigment epithelium-derived factor）又名“色素上皮源因子”或 “色素上皮衍生因子”，为一个多功能性分泌糖蛋白，前人研究表明PEDF 蛋白 具有神经保护性、免疫调节、抑制新生血管生成以及抑制肿瘤恶化等多种功能。 PEDF-R 是PEDF 的受体， 属于PNPLA2 （ Patatin-like phopholipase domain-containing 2 family）蛋白家族的一个新成员，PEDF 蛋白与其结合后会激 活PEDF-R 的磷脂酶A2 活性。本研究中，我们描述了非洲爪蟾PEDF 和PEDF-R 基因的表达图式及其在胚胎发育中的可能功能。RT-PCR 结果显示PEDF 是非母 源性表达，而PEDF-R 则是母源性表达的。原位杂交实验表明它们均在神经系统 中特异表达，但PEDF-R 的表达区域更加广泛，在鳃弓、眼泡和耳泡中也有表达。 通过mRNA 过表达和Morpholino(MO)阻断蛋白合成等手段发现，PEDF 功能获 得和功能缺失后胚胎几乎不受影响。然而PEDF-R 过表达后胚胎向注射一侧弯 曲，TUNEL 凋亡检测实验发现这些胚胎在注射一侧发生了凋亡。这两个基因神 经表达的特异性表明它们可能在早期神经发育中有重要功能。TUNEL 结果暗示 着PEDF-R 可能是一个与凋亡信号通路相关的受体。PEDF 功能获得和缺失并未 导致胚胎明显的表型，这表明PEDF 在非洲爪蟾中可能还存在其他的受体来行使 与PEDF-R 不同功能的途径。 果蝇的vestigial 基因编码一个转录辅助因子，在果蝇中只有一个成员，即 vestigial(vg)基因。在脊椎动物中有四个vestigial 同源基因，即vestigial-like 非洲爪蟾早期胚胎发育中PEDF 和PEDF-R 的功能以及vestigial-like 家族表达图式的研究 2 1,2,3,4_(vgl-1,2,3,4)。Vestigial 蛋白能作为辅助因子与果蝇中的Scalloped(Sd)蛋白 或者哺乳动物中的TEF 蛋白结合成复合体，通过Sd/TEF 蛋白的TEA/ATTS 结构 域与DNA 结合，从而调节下游基因的转录。本研究中，我们克隆了非洲爪蟾 vestigial-like 家族的四个成员，并对其在爪蟾胚胎发育过程中的表达进行研究。 RT-PCR 显示vgl-2 和vgl-3 是合子型表达的，vgl-1、vgl-4 则是母源性表达。原位 杂交显示：vgl-1 主要在神经管背部、耳泡和眼泡中表达；vgl-2 则是在肌肉、第 一二鳃弓、脊索中特异表达；vgl-3 神经胚时期在后脑有强的表达信号，从神经 胚后期到尾芽期后脑部位的表达几乎消失了，而在胚胎的头部以及神经管中开始 有微弱的表达；vgl-4 的表达较广泛，在神经管、眼泡、耳泡、肌肉以及脊索中 均有表达。在爪蟾中这四个成员的表达图式各不相同，提示它们有可能与其行使 组织特异性基因调控的功能相关，上述结果将有助于对vestigial-like 家族基因在 胚胎发育中的功能研究。

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

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.

三叶木通花发育相关基因的结构、功能和进化研究

花是被子植物最关键的创新（innovation）性状。在被子植物的不同类群中，其形态多种多样，尤其以基部真双子叶植物的花形态最为丰富。大量的系统发育分析表明，在核心真双子叶植物起源之前，几个与花发育相关的MADS-box基因亚家族均发生了大尺度的基因重复事件。因此，在被子植物的不同物种中，花发育相关基因的组成并不相同，并且它们经历了不同的进化历史，这意味着这些基因可能以不同的方式调控花的发育。基部真双子叶植物，作为基部被子植物和核心真双子叶植物之间的过渡类群，对于我们理解被子植物花的进化，揭示核心真双子叶植物花的起源以及基部真双子叶植物花多样性分化的分子机制非常重要。本文以基部真双子叶植物三叶木通为研究材料，着重进行了以下研究工作： 1. 花器官发生过程的观察。三叶木通的花为雌雄同序的单性花。而且，根据成熟花的形态，三叶木通的雌花和雄花都只有一轮花被器官，即三个花瓣状的萼片。扫描电镜的观察结果表明：1）在花器官的发生和发育过程中，在萼片和雄蕊原基之间，确实没有花瓣原基或另一轮萼片原基发生。2）雌花和雄花都是以两性花的方式发生发育的。3）单性花是由于在花发育的最后阶段，雌花中雄蕊或者雄花中心皮的退化而产生的。 2. 花发育相关基因的克隆。应用5’/3’ RACE的方法，我们从三叶木通不同发育阶段的混合花芽中共分离到九个与花发育相关的MADS-box基因： AktFL1、AktFL2、AktAP3_1、AktAP3_2、AktAP3_3、AktPI、AktAG1、AktAG2和AktSEP3。 3. A类MADS-box基因的进化。由于A类基因在进化过程中序列结构的改变，再加上取样的限制，使得A类基因间的进化历史一直不能被很好的理解。因此，本文对A类基因的研究从构建该基因亚家族的系统发育树开始。主要结果如下：1）通过扩大在基部真双子叶植物和被子植物其它重要类群的取样，我们的系统发育树基本上反映了现存被子植物的系统发育关系。2）核心真双子叶植物的A类基因由三个分支组成：euFUL、euAP1和AGL79，它们是通过发生在核心真双子叶植物起源之前的两次几乎同时的基因重复事件产生的。3）在基部真双子叶植物中，山龙眼目、毛茛目和黄杨科的A类基因各形成一支。而且，在这些类群内，发生了多次小尺度的独立的基因重复事件。4）来自单子叶植物的FUL-like基因明显地构成一个单系，并且包括三个分支：OsAMDS14、OsMADS15和OsMADS18。它们是由于两次不连续的基因重复事件产生的。5）不同类型的A类基因产物在C末端拥有不同的保守基元。6）从基因组结构上看，所有的A类基因都拥有八个外显子和七个内含子。7）通过对三叶木通中两个FUL-like型基因（AktFL1和AktFL2）表达式样的观察，我们发现它们在叶原基和发育早期的花原基以及发育着的花器官中都有表达。此外，A类基因表达式样的进化分析结果表明被子植物中该类基因的祖先可能具有广泛的功能，既在营养器官中表达又在生殖器官中表达 。 4. B类基因表达式样的保守性和多样性。通过对B类基因的系统发育和表达式样分析，得到以下结果：1）三叶木通中的三个paleoAP3基因是通过两次基因重复事件产生的。2）在木通科或木通属内，PI型基因并没有发生基因重复事件。3）RT-PCR结果表明，AktAP3_1在雌花中的表达量比雄花中高，而AktAP3_2则在雄花中的表达量比雌花中高。AktAP3_3和AktPI在雌花和雄花中的表达水平相似。4）原位杂交分析显示这些基因在发育着的雄蕊和心皮中表达。此外，AktAP3_3和AktPI还在萼片中表达，可能参与花瓣状萼片的发育。 5. 三叶木通C/D和E类基因的序列结构和表达分析。通过序列结构分析，我们发现，与其它被子植物AG同源基因编码的MADS-domain蛋白一样，AktAG1和AktAG2在MADS结构域的N末端都拥有一段氨基酸序列的延伸，AktAG1为20个氨基酸;AktAG2为7个氨基酸。原位杂交分析表明AktAG1和AktAG2主要在发育着的雄蕊和心皮中表达，说明它们具有决定生殖器官发育这一保守的功能。 AktSEP3属于AGL9型的E类基因。该基因在所有花器官中都有表达，说明和其它被子植物的E类基因一样，AktSEP3在三叶木通中对于所有花器官的发育都是必需的。 6. 各类MADS-domain蛋白间的相互作用。在前面工作的基础上，我们首次对三叶木通中上述MADS-domain蛋白间的作用方式进行了研究。酵母双杂交结果表明：1）AktSEP3的C末端具有转录激活功能。2）三个AktAP3蛋白与AktPI蛋白都能够形成异源二聚体，但是它们之间的作用能力并不相同。3）AktSEP3蛋白可以与AktFL1、AktPI、AktAG1和AktAG2形成异源二聚体，充分体现了E类基因产物作用式样的保守性。4）AktFL1与AktPI、AktSEP3和AktAG2也能形成异源二聚体，这与核心真双子叶植物的euFUL型蛋白在作用式样上是非常相似的。 综合以上结果，我们探讨了三叶木通花发育的分子机制。在三叶木通的三轮花器官中，与拟南芥等模式植物相似的是：E类（AktSEP3）基因在每一轮花器官中都起作用;此外，A类（AktFL1）和B类（AktAP3_3和AktPI）基因在花瓣状的萼片中有不同程度的表达，类似于拟南芥的第二轮;B类（AktAP3_1、AktAP3_2、AktAP3_3和AktPI）和C/D类（AktAG1和AktAG2）基因在雄蕊的发育过程中起作用;C/D类（AktAG1和AktAG2）基因对心皮的发育起作用。与拟 南芥等模式植物不同的是：1）虽然原位杂交分析表明，AktFL1、AktAP3_3、AktPI和AktSEP3都在花瓣状的萼片中有 不同程度的表达，但是它们的蛋白质产物AktFL1与AktSEP3和AktAP3_3与AktPI都只能形成较弱的异源二聚体。而 且，根据我们的研究结果，在三叶木通中没有找到euAP1型的A类基因，只有两个FUL-like型的A类基因。它们的功能 与核心真双子叶植物中的euFUL型基因相似。因此，AktFL1很可能与其它调控因子共同作用负责花分生组织的形成;AktFL1/AktAG2则可能在花发育的后期起作用。那么，三叶木通花瓣状萼片的发育是否需要AktFL1/AktSEP3和 AktAP3_3/AktPI的参与，还是另有其它转录因子的参与，仍然需要更深入的研究。2）虽然在三叶木通中，雄蕊的发 育同样需要B、C/D和E类基因的参与，但是由于小尺度的基因重复事件，在该物种中只拥有三个paleoAP3型基因，而没有euAP3型基因。而且，由于复制拷贝间的亚功能化，AktAP3_1/AktPI主要参与雌花的发育过程;而AktAP3_2/AktPI主要参与雄花的发育过程。