A taxonomic revision of the genus Pseudostegana Okada, 1978 (Diptera, Drosophilidae)

The genus Pseudostegana is revised, with descriptions of 20 new species from Southeast Asia: P. angustifasciata Chen and Wang, sp. n., P. atrofrons Chen and Toda, sp. n., P. bifasciata Chen and Toda, sp. n., P. bisetosa Chen and Toda, sp. n., P. curvata Chen and Toda, sp. n., P. dactylis Chen and Toda, sp. n., P. dolichopoda Chen and Wang, sp. n., P. hamata Chen and Toda, sp. n., P. latifasciata Chen and Toda, sp. n., P. leptoptera Chen and Toda, sp. n., P. melanogaster Chen and Toda, sp. n., P. melanopogonias Chen and Toda, sp. n., P. myrmecoformis Chen and Toda, sp. n., P. nitidifrons Chen and Wang, sp. n., P. nitidiventris Chen and Toda, sp. n., P. orbicapitata Chen and Toda, sp. n., P. oxycephala Chen and Toda, sp. n., P. pallidimaculata Chen and Wang, sp. n., P. philoga Chen and Wang, sp. n. and P xanthoptera Chen and Wang, sp. n. On the basis of the wing patterns, six species-groups are established: the atrofrons, grandipalpis, fleximediata, javana, latiparma and zonaria groups. A key to the all species of this genus is provided.

Comparative Expression of Zebrafish Lats1 and Lats2 and Their Implication in Gastrulation Movements

Large tumor suppressor (Lats) is a Ser/Thr kinase, and it presents an important function in tumor suppression. lats was originally identified in Drosophila and recently in mammals. In mammals, it contains two homologues, lats1 and lats2. In the present study, lats1 and lats2 were characterized from zebrafish (Danio rerio), which is the first report of lats in a nonmammalian vertebrate. The primary structure, genomic organization, and phylogenesis of lats from different species were studied, and the results suggest that lats1 is the direct descendant of invertebrate lats, whereas lats2 is formed by genome duplication. In zebrafish, both lats genes are maternally expressed, while they show distinctly different expression profiles during gastrulation. lats1 is almost ubiquitously expressed through development, and lats2 is more prominently expressed in the non-neural ectoderm region of zebrafish gastrula. Most intriguingly, as revealed by cell tracing and gene expression analysis, morpholino-mediated knockdown of either lats1 or lats2 led to obvious defects of cell migration in gastrulation, indicating the functional significance of lats in gastrulation movements. Developmental Dynamics 238:28502859, 2009. (C) 2009 Wiley-Liss, Inc.

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.

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。

转座子Minos介导的果蝇转基因平台构建及水稻高表达启动子的克隆 任娟 指

本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶所在的质粒。转座酶在37度条件诱导下进行表达，协助Minos完成转座过程。在转基因果蝇的阳性筛选中，我们利用眼睛特异表达的绿色荧光蛋作为选择标记。并且，我们通过PCR实验进一步验证了转基因果蝇的真实性。本研究中，我们对转基因实验条件进行了初步优化。我们通过对黑腹果蝇白眼突变品系W1118和D. yakuba注射后胚胎进行保湿，对D. yakuba注射胚胎进行非退壳处理。在改进条件下W1118和D. yakuba的存活率分别为10%和3%左右。通过筛选转基因阳性果蝇，我们得出Minos在W1118和D. yakuba中的转座效率分别在32%和20%左右。我们的实验结果再一次证实了Minos在果蝇D. melanogaster中可行性。同时，该工作也初步完成了在果蝇D. yakuba 中的第一次Minos介导的转基因实验，为新基因的跨物种功能研究奠定了实验基础。在未来的工作计划中，我们将采用Minos转基因系统，把实验室目前研究的黑腹果蝇新基因导入其它物种果蝇进行功能研究。 水稻是一种重要的世界粮食作物，世界上过半的人口以水稻为主食。水稻相对别的粮食作物来讲具有较小的基因组，并且拥有较好的基因组注释，是一种理想的单子叶模式生物。植物转基因技术的发展推动着水稻功能基因组学的研究，目前水稻的转基因技术主要依赖于土壤细菌农杆菌（Agrobacterium tumefaciens）T-DNA介导的外源基因染色体插入。在自然状态下，农杆菌的T-DNA位于Ti致瘤质粒当中。它包括了一些转座元件和一些帮助T-DNA转座的毒性蛋白基因和调节基因。由于Ti质粒上的T-DNA太长，并且没有太多的酶切位点，因此自然状态的T-DNA不适合进行转基因实验。为了方便T-DNA的实际应用，研究人员创立了双载体转基因系统。T-DNA转座区被分离到出Ti载体，并且装载到另外一个适合实验操作的质粒当中，而毒性蛋白表达基因等则保留在Ti质粒上。因此，在进行T-DNA介导的转基因实验时，需要同时存在T-DNA载体和Ti质粒。 本文以“水稻注释计划数据库RAP-DB”的表达数据为参考，选择了60个高表达基因的启动子区域进行克隆。通过对T-DNA载体pCAMBIA1301 进行改造，去掉其原来的35S启动子，将预测的基因启动子克隆到该载体中并与报告基 摘要 因GUS 基因融合。通过分子克隆实验，我们得到了45个高表达基因的启动子载体。最终，为了测试这45个启动子的启动效率，我们会将它们转化到水稻愈伤组织中通过启动子融合的GUS基于表达情况来判断我们启动子的启动效率。

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.

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.

Expression of a twist-related gene, Bbtwist, during the development of a lancelet species and its relation to cephalochordate anterior structures

Mesoderm formation plays a crucial role in the establishment of the chordate body plan. In this regard, lancelet embryos develop structures such as the anteriorly extended notochord and the lateral divertecula in their anterior body. To elucidate the developmental basis of these structures, we examined the expression pattern of a lancelet twist-related gene, Bbtwist, from the late gastrula to larval stages. In late-gastrula embryos, the transcripts of Bbtwist were detected in the presumptive first pair of somites and the middorsal wall of the primitive gut. The expression of Bbtwist was then upregulated in the lateral wall of somites and the notochord. At the late-neurula stage, it was also expressed in the anterior wall of the primitive gut, as well as in the evaginating lateral diverticula. No signal was detected in the left lateral diverticulum when it was separated from the gut, while in the right one, the gene was expressed later during the formation of the head coelom in knife-shaped larvae, and in the anterior part of the notochord in the same larvae. In 36-h larvae, only faint expression was detected in the differentiating notochordal and paraxial mesoderm in the caudal region. These expression patterns suggest that Bbtwist is involved in early differentiation of mesodermal subsets as seen in Drosophila and vertebrates. The expression in the anterior notochord may be related to its anterior expansion. The expression in the anterior wall of the primitive gut and its derivative, the lateral diverticula, suggests that lancelets share the capability to produce a mesodermal population from the tip of the primitive gut with nonchordate deuterostome embryos. (C) 1998 Academic Press.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.