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.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

A conserved family of doublesex-related genes from fishes

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates. (C) 2002 Wiley-Liss, Inc.

A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression

Identifcation of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. Here we report the sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila, zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrfish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibited dlx2 expression in the ventral forebrain. Our studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression. (C) 2001 Academic Press.

果蝇 virilis section 的起源和系统发育研究

果蝇 virilis section(Hsu 1949)自建立以来其合理性在果蝇系统发育中还没有得到 检验。本文以线粒体烟酰胺腺嘌呤二核苷酸脱氢酶第二亚单位基因(ND2)全序列和细 胞色素氧化酶I 基因(COI)部分片段以及核中乙醇脱氢酶基因Adh 的编码区为遗传标 记，对virilis section 内61 个物种(13 个未发表新种),共计117 个个体进行了序列测 定。通过对单个基因和合并数据集进行简约分析，以及对合并数据集进行邻接法分析 和贝叶斯分析，本研究试图对以下问题进行探讨：(1)果蝇virilis section 的合理性。 (2)各种组间的系统关系。(3)种组内部各物种间的系统关系。(4)果蝇virilis section 的进化历史。现将主要结果总结如下： 1)分子系统学研究显示果蝇virilis section 为一个紧密相关的进化簇，为果蝇virilis section 的合理性从分子遗传学的角度提供了证据。 2)系统发育分析支持果蝇polychaeta 种组为单系群，该种组与未归类物种D. fluvialis 具有较近的亲缘关系。它们形成virilis section 中早期分化出的谱系。robusta 种组、melanica 种组、quadrisetata 种组以及新种组间关系较近。其中，robusta 种亚 组和melanica 种组关系较近；quadrisetata 种组和新种组形成姐妹群，它们形成的谱 系与robusta 种组内的okadai 种亚组关系紧密。 3) polychaeta 种组内部分为两枝，姐妹种D. polychaeta 和D. asper 组成其中一枝， 杂交实验显示它们具有非对称的交配前生殖隔离。另一枝中D. latifshahi 和D. daruma 显示了较近的亲缘关系。非洲物种D. hirtipes 与采自西双版纳的D. polychaeta X 关系 较近，提示它们可能有共同的起源。 4)合并数据集支持D. angor 种组为单系群，其中D. angor A 和D. velox 的姐妹种 关系得到较高的支持。但是该种组内部的其它物种间关系还需要进一步研究。 5) 分子系统发育分析结果清楚地表明robusta 种组属于多系发生。该种组被分为 三个种亚组：okadai 种亚组，robusta 种亚组和lacertosa 种亚组。在lacertosa 种亚组 内，D. bai 与其它成员相对远缘。okadai 种亚组的物种由于具有2n = 12 条染色体，且X 染色体为棒状，因而被认为是robusta 种组中较早分化出来的类群。但是我们的 分子数据并不支持这种观点。D. moriwakii 最初被描述为robusta 种组物种，后被订正 至melanica 种组。我们的系统发育分析显示该种与robusta 种亚组具有较近的关系， 而与melanica 种组物种的关系相对较远。除D. moriwakii 外，melanica 种组新旧大陆 物种各自形成单系。在系统树上，中国品系D. tsigana 与D. longiserrata 关系较近， 而与日本品系D. tsigana 的关系较远。杂交实验结果显示D. tsigana 中国品系和日本 品系间存在不对称的交配倾向，表明该种不同的地理群体间可能正处于分化阶段，而 中国品系则有可能已经演化为不同的物种。 6)果蝇quadrisetata 种组为单系发生，其内部分为两个明显的亚世系。第一个亚 世系包括D. sp T，D. barutani，D. potamophila 和D. spIZU；另一个亚世系包括D. beppui，D. karakasa，D. quadrisetata，D. multidentata，D. perlucida 和D. pilosa，其 中D. beppui 为该谱系中最早分化出的物种。 通过估计谱系间的分歧时间，本文推测果蝇virilis section 的祖先大约于中新世早 期起源于热带地区，virilis section 内许多物种可能栖息在旧大陆的低纬度地区，然后 通过适应性辐射扩散到各地。几乎所有的新大陆virilis section 物种是旧大陆物种通过 白令陆桥迁移到新大陆而演化形成的。

果蝇virillis section的起源和系统发育研究

果蝇vlfrlflisseetion（Hsu1949）自建立以来其合理性在果蝇系统发育中还没有得到检验。本文以线粒体烟酞胺腺嘌吟二核昔酸脱氢酶第二亚单位基因（ND2）全序列和细胞色素氧化酶I基因（COl）部分片段以及核中乙醇脱氢酶基因Adh的编码区为遗传标记，对viriltsseetion内61个物种（13个未发表新种），共计117个个体进行了序列测定。通过对单个基因和合并数据集进行简约分析，以及对合并数据集进行邻接法分析和贝叶斯分析，本研究试图对以下问题进行探讨：（1）果蝇virilisseetion的合理性。（2）各种组间的系统关系。（3）种组内部各物种间的系统关系。（4）果蝇virilissection的进化历史。现将主要结果总结如下：1）分子系统学研究显示果蝇virilissection为一个紧密相关的进化簇，为果蝇virilissection的合理性从分子遗传学的角度提供了证据。2）系统发育分析支持果蝇polychaeta种组为单系群，该种组与未归类物种D．Uvl'alis具有较近的亲缘关系。它们形成virilissection中早期分化出的谱系。robusta种组、melanica种组、quadrisetata种组以及新种组间关系较近。其中，robusta种亚组和melanica种组关系较近；quadrisetata种组和新种组形成姐妹群，它们形成的谱系与robusta种组内的。加dai种亚组关系紧密。3）polychaeta种组内部分为两枝，姐妹种D．polychaeta和D.asPer组成其中一枝，杂交实验显示它们具有非对称的交配前生殖隔离。另一枝中D.Iatshahi和D.daruma显示了较近的亲缘关系。非洲物种D．hirtiPes与采自西双版纳的D．polychaetaX关系较近，提示它们可能有共同的起源。4）合并数据集支持D．angor种组为单系群，其中D．angorA和D.velox的姐妹种关系得到较高的支持。但是该种组内部的其它物种间关系还需要进一步研究。5）分子系统发育分析结果清楚地表明robusta种组属于多系发生。该种组被分为三个种亚组：okadai种亚组，robsta种亚组和lacertosa种亚组。在laCertosa种亚组内，D.bai与其它成员相对远缘。okadai种亚组的物种由于具有2n=12条染色体，且X染色体为棒状，因而被认为是robusta种组中较早分化出来的类群。但是我们的分子数据并不支持这种观点。D.moriwakii最初被描述为robusta种组物种，后被订正至melanica种组。我们的系统发育分析显示该种与robsta种亚组具有较近的关系，而与melanica种组物种的关系相对较远。除D．moriwakii外，melanica种组新旧大陆物种各自形成单系。在系统树上，中国品系D．tsigana与D．longiserrata关系较近，而与日本品系D．tsigana的关系较远。杂交实验结果显示D．tsigana中国品系和日本品系间存在不对称的交配倾向，表明该种不同的地理群体间可能正处于分化阶段，而中国品系则有可能已经演化为不同的物种。6）果蝇quadrisetata种组为单系发生，其内部分为两个明显的亚世系。第一个亚世系包括；另，，个亚世系包括D．，其中D．beppui为该谱系中最早分化出韵物种。通过估计谱系间的分歧时间，本文推测果蝇virilissection的祖先大约于中新世早期起源于热带地区，virilissection内许多物种可能栖息在旧大陆的低纬度地区，然后通过适应性辐射扩散到各地。几乎所有的新大陆virilissection物种是旧大陆物种通过自令陆桥迁移到新大陆而演化形成的。

哺乳动物新Y 染色体的退化和果蝇新基因起源模式的研究

进化生物学得益于近代分子生物学和当代基因组学的发展，已经脱 离了自达尔文时代起博物学式的观察和思辨性的研究状态。很多古老而又 经典的问题，因为在一些年轻的进化系统中的研究，绽放出其背后深刻的 机制。在本工作中，我们通过在模式物种果蝇和珍稀动物黑麂中的研究， 揭示了有关遗传的基本单位-- 基因是如何起源和消亡的，以及这些重要过 程背后的规律。 决定人类雄性的Y 染色体起源于一亿六千万年前X 染色体的同源 染色体。但现今Y 染色体上的基因数目仅仅是X 染色体的百分之一左 右。如此巨大的数目差异，是由于Y 染色体和X 染色体之间重组抑制以 后，大量的Y 染色体基因发生退化消亡所致。 由于哺乳动物的Y 染色体 大都非常古老，Y 退化的过程和机制一直以来无法得以深入研究。 在本工 作的前半部分，我们首次在中国特有的珍稀鹿科动物黑麂中报道鉴定了一 对行为和模式类似人类性染色体的常染色体。这对“新性染色体”(neosex) 仅仅起源于50 万年以内，由于雄性特异的染色体倒位，致使数以千计 的基因像Y 染色体连锁的基因那样，无法与其等位基因重组。对23 个新 Y 染色(neo-Y)体连锁的基因25kb 的蛋白编码区和它们35kb 的非编码区的 序列分析发现，与其他可重组区域相比，这些基因的遗传多态性显著降 低，并积累了改变氨基酸的有害突变。我们还首次用体内表达试验证明Y 染色体的基因在其顺式调控区域也发生了退化。这些积累在启动子或者非 翻译区域(UTR)的有害突变，将扰乱Y 染色体上基因的正常表达，并进一 步促进退化过程和剂量补偿效应以单个基因(gene-by-gene)的模式进化。 本论文的另外一部分工作主要研究了果蝇中新基因起源的总体模式 问题。对遗传新元件如何起源的兴趣，最早可以追溯到达尔文。近年来通 过对“年轻基因”的案例研究，我们已经知道通过基因重复，逆转座，水 平迁移和从头起源等机制可以产生新基因。但这些机制在全基因组水平对 新基因起源的贡献各自如何，以及以非编码区从头起源合成一个新的基因 是否普遍等重要问题一直未得到解答。我们利用比较基因组的手段，在6个果蝇全基因组中，通过12017 个黑腹果蝇基因序列，鉴定刻画了超过 300 个起源于不同时间点的新基因。我们对这些新基因的序列，结构和表 达模式的分析发现，串联重复在产生年轻的新基因过程中占了主导地位 (超过80%)。但是最后固定在群体内，有功能的新基因主要(44.1%)是散在 重复的形式。我们惊奇地发现非编码区从头起源的基因在新基因的起源过 程中也扮演了重要角色，产生了超过10%的有功能的新基因，并且大部分 都进化出了睾丸特异的表达模式。有大约30%的新基因通过招募其他基因 的编码区或者重复元件，形成了新的嵌合结构，暗示它们可能获得了新的 功能。最后，我们估计在果蝇中，每百万年将产生5 至11 个有功能的新 基因。

天然免疫相关分子的进化

本论文结合功能研究和进化遗传学方法对动物天然免疫（innate immunity）相关分子的进化历程进行深入研究。受体对病原微生物的识别是天然免疫系统发挥功能的基础。作为模式识别受体（pattern recognition receptor, PRR），果蝇肽聚糖识别蛋白SD（PGRP-SD）在识别革兰氏阳性细菌的过程中发挥了重要作用。针对已有的黑腹果蝇（Drosophila melanogaster）群体数据，我们发现PGRP-SD在群体中存在2类高频的等位基因（分别为等位基因1和等位基因2）。以D. simulans为外群，我们追溯了黑腹果蝇2类等位基因上氨基酸的变化。这些氨基酸的结构特征和在蛋白质上所处的位置提示这2类等位基因在功能方面可能存在分化。通过功能研究的方法，我们发现在黑腹果蝇中该基因功能方面发生了显著的变化。等位基因2在有微生物时能激活天然免疫反应，但等位基因1的转基因果蝇成虫只要有外伤即便没有微生物的情况下即能激发天然免疫反应，而带有等位基因2果蝇成虫则不具有该功能。这一结果提示我们，发生在该等位基因上的氨基酸变化导致了其识别功能的变化。与推导的祖先基因相比，等位基因1发生了一个氨基酸的变化，因此导致其功能从识别细菌细胞壁组分肽聚糖转变为一未知的自身组分，即从病原相关分子模式（pathogen-associated molecular pattern，PAMP）识别受体转变为损伤相关识别模式（damage-associated molecular pattern, DAMP）识别受体。通过这一功能变化， 果蝇成虫可以通过仅识别自身损伤即可激活相应的免疫反应，对后续可能侵入的微生物进行杀伤。已有研究结果显示，微生物在进化过程中已经形成针对DAMP和PAMP规避策略。上述2类等位基因的同时存在能使黑腹果蝇同时具备两个机制，更加充分地抵抗病原微生物的入侵。结合功能研究和针对自然群体的群体遗传学分析，我们认为在黑腹果蝇群体中以高频共存的2类PGRP-SD等位基因可能可能受到了平衡选择（balancing selection）作用。上述工作主要研究了天然免疫系统识别受体的进化。而本论文的另一部分则主要针对天然免疫系统的效应分子（effector）进行了研究。作为重要的效应分子，抗菌肽在杀菌方面发挥着最为直接的作用。因此，研究抗菌肽的进化对于探索天然免疫系统的进化具有重要意义。本研究以两栖类动物大蹼铃蟾抗菌肽基因家族为例，通过对分别来自2个大蹼铃蟾个体的皮肤cDNA文库进行测序，我们鉴别出56个不同的抗菌肽cDNA序列。每一个cDNA均编码2个不同的抗菌肽，maximin 和maximin H。基于针对这些cDNA序列的分析，我们发现2类抗菌肽编码序列的非同义替代率均高于同义替代率，呈现高度分化的特征。但是，在信号肽和其它非抗菌肽编码区域并没有发现这种情况。这一结果提示抗菌肽可能受到超显性选择（overdominent selection, 即平衡选择）的影响。同时，我们分别从皮肤和肝脏克隆基因了7个抗菌肽的基因组编码序列并进行了测序。这些从不同组织获得的抗菌肽在各个编码序列中均存在序列的差异的同时呈现了相同的结构。这一结果提示不同抗菌肽间的差异不太可能来自于体细胞突变而是快速序列进化的结果。通过构建来自于同一个体的抗菌肽的不同编码区的基因树，我们发现结构域重排（domain shuffling）和/或基因转换（gene conversion）在这些抗菌肽的进化历程中发挥作用。

小鼠Bicc1 基因的克隆、Bicc1 多克隆抗体制备及蛋白定位与表达的研究

双尾-C 基因 (Bicaudal-C)首先在果蝇（Drosophila melanogaster）中发现，其功能丧失导致果蝇胚胎滤泡细胞的错误迁移、头部的缺失和双尾结构的形成。后来发现多个物种都含有Bicaudal-C 的同源基因，其中小鼠中的同源基因Bicc1 的缺失导致小鼠产生肾脏等脏器的病变，其症状与人类多囊肾疾病高度相似，但其具体机制还不清楚。本研究以小鼠肾脏组织总RNA 为模板体外反转录为cDNA，通过分段巢式 PCR 及酶切连接的方法获得了全长约3Kb 的小鼠Bicc1 cDNA 序列。根据生物信息学分析全长的Bicc1 蛋白，选择两个免疫原性较好的区段作为抗原位点构建相应的原核表达载体；IPTG 诱导表达并纯化融合蛋白，制备两种兔抗Bicc1 蛋白多克隆抗体，并通过Western blot 证实这两种抗体具有高度特异性。用细胞免疫荧光方法及免疫组织化学方法对该蛋白的定位做了一些初步研究。发现Bicc1 蛋白定位于体外培养的小鼠肾细胞的细胞质内，并在胚胎发育于期表达仅在心脏，后来逐步地在各个组织器官内出现，并在出生后的小鼠体内表达稳定。Bicc1 mDNA 也表达于多个器官内，并且在肾脏中有明显较高的表达量。找到了的两个针对Bicc1 基因的RNAi 的序列，通过荧光强度变化和Western blot 均证明这两个序列能明显降低Bicc1 蛋白在体外培养细胞中的表达水平，为下一步建立稳定的细胞株奠定了良好的基础。

横断山区果蝇科的区系分类及伊米果蝇种组各种亚组间的系统发育关系研究

本文新种部分，记述了分布在横断山地区、台湾以及苏门答腊、菲律宾等地果蝇科的新种55种，异物同名3种。在果蝇属果蝇亚属的伊米果蝇种组建立了一个新的种复组－弯头果蝇种亚组(Drosophila curviceps species subgroup)；在quadrilineata种亚组内建立了一个新的种复组－背条果蝇种复组(Drosophila notostriata species-complex)。对横断山地区的果蝇进行研究，计有119个种，主要由东洋种、古北种、特有种和广布种组成；对其渊源关系进行分析。运用分支分类学的观点和方法对伊米果蝇种组中5个种亚组间的系统发育进行研究显示，hypocausta种亚组最原始，位地分支图的最底部，是外群向伊米果蝇种组过渡的一个类群，进化中心在热带的苏门答腊及其附近；其次分化的immigrans种亚组其进化中心仍在热带的苏门答腊及其附近；但该亚组的特种逐渐向北扩散，在中国云南的西双版纳及其附近地区形成次级进化中心。伊米果蝇种组总的进化方向是从热带向温带的逐渐演化。

从基因组角度探讨果蝇蛋白质的进化

蛋白质进化的驱动力是分子进化中最基本的问题之一，研究者们早已在群体 遗传学研究中做出了许多模型和推测。基因组时代的到来使得证实这些经典理论 成为可能果蝇12 个基因组测序计划产生了大量新的数据，通过分析这些新的数 据，可以获得对果蝇蛋白质进化的更深入认识，也可以为分子进化研究的许多经 典理论提供验证。 我们利用黑腹果蝇种组的六个基因组，结合比较基因组学和功能基因组学的 研究手段，对黑腹果蝇种组中蛋白质的进化速率与许多可能影响蛋白质进化的功 能基因组学参数进行了统计学分析，从全基因组水平上探讨了果蝇蛋白质的进 化。 研究结果表明，表达特异性是决定果蝇蛋白质进化的最显著因素，广泛表达 的持家基因具有更低的进化速率，而表达量尽管与蛋白质进化呈负相关，但是这 个效应并不强，表达对蛋白质进化的影响与密码子偏好性和基因的功能多样性无 关，可能是由翻译后选择导致的。基因的功能重要性和基因的功能密度都对蛋白 质的进化有显著的影响，二者之间存在一定的联系，但是基因的功能密度对蛋白 质进化的影响要大于基因的功能重要性。蛋白质长度受到表达量选择，高表达的 基因往往具有更短的蛋白长度，因此，高表达蛋白的蛋白质长度与蛋白质进化速 率不相关，但是蛋白质长度在非高表达基因中对蛋白质进化存在显著的影响，其 机制可以部分的由基因的功能密度来解释，更确切的机制还需进一步研究。第一 个内含子的长度与蛋白质的进化速率显著相关，可能是由于第一个内含子中存在 较多的调控序列导致。基因重复的固定机制主要是高复杂度基因发生重复后的亚 功能化，而功能补偿效应和剂量平衡效应并不重要。基因的重组率在全基因组水 平上与蛋白质进化速率显著负相关，可能是因为基因组中大多数蛋白并不参与到 正选择的过程当中所致。 本研究系统的分析了影响果蝇蛋白质进化速率的各种功能基因组学参数的 效应，除了重新分析了过往研究中提到的基因表达水平，蛋白质相互作用，蛋白质长度这些参数在果蝇蛋白质进化中的影响，本工作还分析了许多新的参数，包 括表达特异度，内含子长度，基因功能重要性，基因功能密度，基因重复，基因 重组等。我们的工作不仅进一步证实了许多在进化基因组学研究中被广泛接受的 理论，还获得了一些与过去研究不同的结果，例如，果蝇蛋白质长度与蛋白质进 化速率呈负相关，第一个内含子在蛋白质进化中有很重要的作用等。

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

P>NF-kappa B is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-kappa B-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor kappa B (I kappa B)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-kappa Bs and phylogenetic analysis suggested that EsRelish was a member of the NF-kappa B family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

First molluscan TNFR homologue in Zhikong scallop: Molecular characterization and expression analysis

Tumor necrosis factor receptors (TNFRs) are a superfamily of proteins characterized by the unique cysteine-rich domain (CRD) and their important roles in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The first member of the molluscan TNFR family, designated as CfTNFR, was identified from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTNFR was of 1334 bp, consisting of a 5' UTR of 17 bp, a 3'UTR of 69 by with a poly (A) tail, and an open reading frame (ORE) of 1248 by encoding a polypeptide of 415 amino acids with a theoretical isoelectric point of 8.33 and predicted molecular weight of 47.07 kDa. There were a signal peptide, a CRD, a transmembrane region and a death domain in the deduced amino acid sequence of CfTNFR, suggesting that it was a typical type 1 membrane protein. The high identities (22-40%) of CfTNFR with other TNFR superfamily members indicated that CfTNFR should be a member of TNFR superfamily, and moreover, it should be the first death domain-containing TNFR found in invertebrates. Phylogenetic analysis revealed that CfTNFR was closely related to TNFR-like proteins from Strongylocentrotus purpuratus, Drosophila melanogaster and Ciona intestinalis, and they formed a separate branch apart from vertebrate TNFRs. The spatial expression of CfTNFR transcripts in healthy and bacteria challenged scallops was examined by quantitative real-time PCR. CfTNFR transcripts could be detected in all tested tissues, including haemocytes, gonad, gill, mantle and hepatopancreas, and significantly up-regulated in the tissues of gonad, gill, mantle and hepatopancreas after Listonella anguillarum challenge, indicating that CfTNFR was constitutive and inducible acute-phase protein involved in immune defence. The present results suggested the existence of the TNFR-like molecules and TNF-TNFR system in low invertebrates, and provided new insights into the role of CfTNFR in scallop innate immune responses to invading microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.

Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35A degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25A degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.

The genomic structure, alternative splicing and immune response of Chlamys farreri thioester-containing protein

MEP is a member of thioester-containing protein (TEP) family found in Zhikong scallop Chlamys farreri and is involved in innate immunity against invading microbes. In the present study, the genomic DNA of CfTEP was cloned and characterized. The genomic DNA sequence of CfTEP consisted of 40 exons and 39 introns spanning 35 kb with all exon-intron junction sequences agreeing with the GT/AG consensus. The genomic organization of CfTEP was similar to human and mouse 0 rather than ciona C3-1 and Drosophila dTEP2. By RT-PCR technique, seven different cDNA variants of CfTEP (designated as CfTEP-A-CfTEP-G) were cloned from scallop gonad. CfTEP-A-CfTEP-F were produced by alternative splicing of six mutually exclusive exons (exons 19-24), respectively, which encoded the highly variable central region. While in CfTEP-G, the deletion of all the six exons introduced a new translation stop site and might trigger nonsense mediated decay (NMD). The mRNA expression and the proportion of the seven CfTEP variant transcripts were examined in the gonad of scallops after bacterial challenge. The fragments containing the highly variable central region of UTEP were amplified by RT-PCR and a 100 positive clones were sequenced randomly. The expression profiles of the seven MEP variants were different and displayed the sex and bacteria dependent manner. In the blank, sea water and Listonella anguillarum challenged subgroups of male scallops, all the transcripts detected were CfTEP-G isoform. In the Micrococcus luteus challenged subgroup, the isoforms expressed and their proportions were CfTEP-F (54%), CfTEP-B (23%), CfTEP-A (10%), CfTEP-C (7%) and CfTEP-E (6%). However, in the gonad of female scallops, only CfTEP-A were found in blank and sea water challenged subgroups. After L anguillarum or M. luteus challenge, four and five isoforms were detected, respectively, with CfTEP-F isoform being the most one in the both subgroups. These results suggested that the evolution of TEP genes was very complex, and that the diverse CfTEP transcripts generated by alternative splicing played an important role as pattern recognition receptors in the innate immune defense of scallops. (C) 2009 Elsevier Ltd. All rights reserved.