Prokaryotic Expression of Amphioxus BRA Protein and Preparation of the Polyclonal Antibody

Brachyury编码的转录调控因子BRA参与脊索动物脊索的分化形成,文昌鱼是最早具有真正脊索的后生动物类群,因此开展文昌鱼Brachyury基; 因功能研究,将有助于揭示脊索的起源与进化。文昌鱼具有2个Brachyury基因:Bra1和Bra2,二者编码的蛋白序列相似度高达93%,缺少有效; 区分二者的特异抗原表位,转录组数据分析表明Bra2表达量显著高于Bra1,进一步对BRA2蛋白序列特征分析发现其N端拥有丰富的潜在抗原决定簇,因; 此本研究选择了Bra2 N端696; bp基因序列所编码的蛋白片段作为制备抗体的抗原蛋白。将该段基因序列克隆重组入pET28a原核表达质粒,经诱导表达获分子量约31; ku的可溶性重组蛋白。通过Ni~(2+)亲和层析柱纯化,得到1.3 g/L高纯度抗原蛋白,用3只ICR小鼠(Mus; musculus)经4轮重组蛋白免疫[剂量50 mug/(只·次)]后获得最高效价(1︰256; 000)的多克隆抗体。Western印迹结果显示,本研究制备的鼠抗文昌鱼BRA多克隆抗体不仅可特异识别重组抗原蛋白,也可高效识别文昌鱼胚胎总蛋白; 中的BRA1和BRA2,为后续深入研究文昌鱼BRA在脊索发育调控中的作用提供了有力的分子工具。Homologues of the T-box gene Brachyury play an essential role in; notochord specification of early embryo development of chordates.; Amphioxuses, also called cephalochordates, represent the most basally; divergent lineage of chordates, being the sister group of urochordates; and vertebrates. It is now generally agreed that amphioxus is the first; animal group to evolve a real sense of notochord in all metazoans, hence; studying Brachyury in amphioxus would shed important insights into the; origin and evolution of notochord. In order to explore the function of; Brachyury in the developmental regulation network of amphioxus, we; generated a mouse anti-amphioxus BRA polyclonal antibody. Amphioxus; possesses two Brachyury homologues genes: Bra1 and Bra2, encoding two; proteins with a sequence similarity of 93% (Fig. 1a). It is very hard to; distinguish specific epitopes between them. The transcriptome data; showed that the expression level of Bra2 was much higher than that of; Bra1 (Fig. 1b). Further analysis of BRA2 sequence feature indicated that; ideal presume antigenic determinants located in the N-terminal of BRA2; (Fig. 1c). Therefore, a 696 bp gene segment was amplified by PCR from; Bra2 N-terminal (Fig. 2a) and inserted into prokaryotic expression; vector pET28a (Fig. 2b). The recombinant plasmid pET28a-Bra2-N was; transformed into Escherichia coli BL21 and induced to express the tagged; protein (Fig. 3a). We obtained a huge amount of soluble recombinant; protein with an expected size (31 ku) (Fig. 3b) and purified the tagged; protein using Ni~(2+) affinity chromatography (Fig. 3c). Three ICR mice; were immunized to generate polyclonal antibodies against amphioxus BRA2; with purified recombinant protein (1.3 g/L). The enzyme-linked; immunosorbent assay (ELISA) showed that the titer of mouse; anti-amphioxus BRA2 antibody was 1︰256 000, with a high sensitivity; (Fig. 4a). Western blot experiments showed that the polyclonal antibody; could not only effectively identify recombinant protein (Fig. 4b) but; also recognized amphioxus BRA1 and BRA2 (Fig. 4c, Fig. 5a, b). In; conclusion, we successfully generated the mouse anti-amphioxus BRA; polyclonal antibody that would be a powerful molecular tool for further; investigating the function of Brachyury in amphioxus.国家自然科学基金项

文昌鱼中一个2A肽介导的多基因表达载体构建

2A肽(P2A)介导的多基因表达载体具有高裂解活性且上、下游基因等摩尔表达等优点,已广泛应用于动物转基因研究.文昌鱼(amphioxus)作为一种新兴的模式动物,尚无应用这种表达载体的报道,为此在pXT7转录系统基础上构建一个P2A介导的多基因表达载体.将体外转录的P2A介导的mRNA注入文昌鱼卵细胞并受精后,经激光共聚焦显微镜和蛋白质免疫印迹(Western blot)检测表明,该mRNA在文昌鱼胚胎中能够高效地翻译和剪切,并且在信号肽的作用下eGFP蛋白定位于细胞核中,而mCherry蛋白定位于细胞膜上,上、下游蛋白间的剪切效率达到91%;进而构建了由文昌鱼热激蛋白基因启动子(BbHsp70)启动,并由P2A介导的多基因表达载体,实验证明其在热诱导和上、下游蛋白剪切方面均达到了预期效果.国家自然科学基金(31372188,31471986,31672246

Progress in the Research on the ABCA Gene Family of Vertebrates

ABC(ATP-binding cassette)基因家族编码膜蛋白,其成员负责多种物质的跨膜运输。基于氨基酸序列的同源性,人的48个ABC成员被分为7个亚家族:ABCA~ABCG。与其他亚家族相比,ABCA基因编码的蛋白具有独特的拓扑结构,并且其家族成员在两栖动物和哺乳动物分化之后各发生过一次大的扩展(expanding)。基因结构分析发现这两次扩展均是通过基因倍增实现的,这些倍增的产物在啮齿目和食肉目中得到保留,而在灵长目中却有一半变成假基因或被删除。ABCA成员主要负责不同组织器官脂类和胆固醇的跨膜运输,部分成员的突变与疾病相关。The ATP-binding cassette(ABC) superfamily encodes membrane proteins that transport many kinds of substrates across membranes.Based on amino acid sequence similarities and phylogeny,48 ABC genes in the human genome were divided into seven subfamilies: ABCA to ABCG.Among them,ABCA transporters have a unique topology and the members of this subfamily had expanded twice by gene duplication after the divergences between amphibian and mammal,respectively. The new duplicated genes are well retained in rodent and carnivore,while half of them became pseudogenes or were lost in primate genome.Transporters of the ABCA subclass were responsible for critical physiological functions in the transmembrane transportation of endogenous lipid and cholesterol substrates.Mutations of ABCA genes are associated with human genetic diseases.基金国家自然科学基金(编号:30470938,30570208);; 福建省自然科学基金(No.D0510002);; 厦门市科技计划重点项目(编号:3502Z20042015)资助~

Generating amphioxus Hedgehog knockout mutants and phenotype analysis

文昌鱼隶属脊索动物门头索动物亚门,是无脊椎动物到脊椎动物的过渡类群,其躯体结构简单,是研究胚胎发育的理想材料。本文以文昌鱼为实验对象,利用TAlEn敲除技术对HEdgEHOg(HH)基因在胚胎发育中的功能进行了研究。在文昌鱼HH基因翻译起始位点下游附近选取TAlEn目标位点,根据此序列组装相应TAlEn重组质粒,体外合成M rnA,向未受精卵注射M rnA后,经体外受精获得f0代胚胎。效率分析显示,靶向该基因的TAlEn M rnA可导致f0代代胚胎在相应基因组区域发生突变的比例为34%。对部分f0个体所产配子筛查发现,TAlEn引起的突变可进入配子,将其中1尾突变类型为8 bP缺失的雄性个体与野生型雌性配对获得f1群体,对f1群体逐尾筛查,从中获得多尾携带8 bP缺失的杂合子;这些杂合子相互配对所产的f2代胚胎,其中约有1/4个体在幼体早期出现躯体前端和尾向下弯曲、脊索前端腹侧的中胚层组织发育不全,不能开口等;随着幼体生长发育,躯体前端和尾部进一步卷曲,口部仍未形成,左右各形成一个口前窝,内柱和鳃裂位于躯体腹侧,最终因无口摄食而死亡。基因型分析发现,上述畸形胚胎均为HH纯合突变体,其与杂合子及野生型比例分布符合孟德尔遗传定律,表明这些发育畸型的特征与HH基因功能缺失有关。The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree.In the present study, we reported a genetically modified amphioxus strain on the Hedgehog(Hh) gene locus using the TALEN method.The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region.Further analysis on the F0 gametic DNA revealed that the mutations had entered into gametes.So, we paired one F0 male carrying an 8 bp deletion with a wild-type(WT) female, and carefully nursed the F1 embryos up to adulthood.We then screened F1 individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F1 offspring.Moreover, our observation on the F2 embryos generated by mating F1 mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening.With the larva growth, deformities(such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake.Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law.WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side.However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis.国家自然科学基金项目(编号:31372188;31101631)资

Wnt signaling pathway and the Evo-Devo of deuterostome axis

动物体轴极性的建立和最初胚轴的形成涉及到一系列信号通路的调控,WnT信号通路是其中一条十分保守的信号通路,并且WnT/β-CATEnIn信号通路中的关键成员早在海绵动物中就有发现,暗示这一信号通路相对于其他信号路径来说可能是最早参与原始后生动物体轴发育的信号通路之一,并且在体轴后端和腹部的发育及命运分化方面发挥着重要作用。近年来,随着体外功能实验体系的建立,人们发现WnT信号通路中很多基因都不同程度地影响了早期胚轴的形成,例如WnT基因、母源性基因β-CATEnIn以及一系列转录因子等。文章首先对参与后生动物体轴发育的WnT基因家族的起源与进化关系做一简要分析,并进一步就经典的WnT/β-CATEnIn通路与后口动物的海胆、文昌鱼、斑马鱼、爪蟾和小鼠等类群体轴极性的建立乃至整个体轴形成方面的研究进展做一综述。A series of signal transduction pathways have been found to regulate the polarity establishment and formation of animal primary body axis.Among them,Wnt signaling pathway is extremely conserved and several key components in the pathway have been identified in the demosponge lineage.This implies that it is one of the earliest pathways involved in the ancestral metazoan axis development and might play an important role in specification and development of posterior and ventral fate of animal axis.Recently,with the establishment of functional experiments in vitro,the body plan formation has been found to be affected,in varying degrees,by many genes in the Wnt signaling pathway,such as members of wnt gene family,maternal gene β-catenin and some transcription factor encoding genes.In this review,we analyzed the evolu-tionary origin of the wnt gene family involved in development of metazoan body plans,and then made a brief review on the roles of canonical Wnt/β-catenin signaling in the polarity establishment and formation of primary body axis in diverse deu-terostomes including sea urchin,amphioxus,zebrafish,frog,and mouse.国家自然科学基金项目(编号:30830023;31071110)资

Research progress of conserved non-coding elements in metazoan

作者简介: 冯俊, 硕士研究生, 专业方向：动物发育遗传学。Tel: 0592-2184427; E-mail: [email protected] 通讯作者: 王义权, 教授, 博士生导师, 研究方向：发育遗传与比较基因组学。E-mail: [email protected] 网络出版时间: 2012-11-21 9:25:09 URL: http://www.cnki.net/kcms/detail/11.1913.R.20121121.0925.001.html[中文文摘]生物体基因组中除了编码序列之外,还存在大量的非编码调控序列。比较基因组学研究发现:脊椎动物、尾索动物、头索动物、果蝇、线虫等基因组中存在保守的非编码调控序列。这些非编码保守元件通常分布在与转录调控发育相关的基因上下游区域,作为基因调控网络核心的一部分,常常在基因表达过程中扮演转录增强子的角色。文章总结了近年来有关后生动物非编码保守元件的发现和主要特点,并进一步就非编码保守元件在大规模基因组倍增之后的演化及其在生物躯体图式进化过程中的影响进行了综述。[英文文摘] In addition to protein coding sequence, the organism genome contains a significant amount of regulatory DNA. Comparative genomics reveals that the organism genomes of vertebrates, tunicate, cephalochordate, flies, and nematodes contain cis-regulatory elements with highly conserved non-coding elements (CNEs). CNEs that cluster around trans-dev genes are part of core gene regulatory networks (GRNs), and usually, they can act as transcriptional enhancers. In this review, we described the identification of CNEs and summarized their key properties across the metazoans, and then discussed the evolution of CNEs after large-scale genome duplication events and the role of CNEs in the evolution of animal body plan.国家自然科学基金项目(编号:30830023;No.31071110);教育部博士点基金(编号:20110121120002)资

Chromosome Preparation and Preliminary Observation of Two Amphioxus Species in Xiamen

文昌鱼的进化地位十分重要,对其染色体的研究在进化和比较基因组学方面有重要意义。然而文昌鱼的染色体制备困难,使研究受到了限制。本文介绍了一种改良的文昌鱼胚胎细胞染色体标本制备方法,以及用文昌鱼成体再生细胞制备染色体,首次获得了文昌鱼体细胞中期染色体标本,并观察了厦门2种文昌鱼的染色体,其中白氏文昌鱼(brAnCHIOSTOMA bElCHErI)二倍体2n=40,日本文昌鱼(b.JAPOnICuM)二倍体2n=36。再次从细胞分类学角度证实白氏文昌鱼和日本文昌鱼作为两个独立物种的分类地位。Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates.Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics.The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research.In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells.Chromosomes of two amphioxus species from Xiamen waters in China are also observed.The diploid chromosome number was found to be 40 in Branchiostoma belcheri, while B.japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.SupportedbygrantsfromNSFC(No.30570208);SRFDPofMinistryofEducation;China(20070384041);TheEducationDepartmentofFujianProvince;China(JB07063

脊椎动物线粒体DNA的基因重排

将GenBank上已公布的321种脊椎动物mtDNA全序列,按纲整理归类,绘制基因排布图并进行比对。 比对结果表明:81个物种的mtDNA中观察到基因重排现象,涉及脊椎动物各纲,其中9个物种同时存在基因顺序 变化和基因倒置现象,所有的基因重排都涉及tRNA的变化。脊椎动物mtDNA基因顺序变化可分为3类:1)邻接 的基因或片段的位置交换;2)接近于控制序列或轻链起始位点的基因或片段的位置变化,有时还伴随着控制序列 的倍增;3)I Q M区域的变化。所有鸟类、蛇类、鳄类和有袋类的mtDNA具有各自独特的基因排列顺序。基因倒 置现象常见于鱼类和哺乳类,且多表现为tRNA从轻链往重链上迁移。本文就这些基因重排现象、发生重排的机制 和mtDNA基因重排在系统发生研究中的应用做一简要概述。国家自然科学基金(编号:30170505,30470938);; 教育部骨干教师资助计划(编号:GG 180 21002403 1740);; 教育部留学回国人员启动基金资助~

Mass Cultivation of Feed Organism for Amphioxus in Laboratory

运用实验生态学方法研究了海产动物重要饵料生物———叉鞭金藻(Dicrateriasp.)在室内大量培养的条件和方法。结果表明,营养盐浓度对叉鞭金藻的生长没有明显影响,以1倍浓度的F/2培养基最佳;水温21~27℃的范围都适合叉鞭金藻的培养,27℃下的生长速度和种群最终密度都高于其他温度;在实际应用中,接种密度以10×104~30×104个mL为最佳;在收获方式上,一次性培养与半连续培养的最终收获量没有显著差别,但半连续培养较一次性培养更有利于操作和减轻工作量。We have studied the condition and method for indoor mass cultivation of Dicrateria sp.,which is an important species for marine animal feeding.The results showed that concentration of nutrition salts did not obviously affect it's growth,and 1×F/2 medium would be the optimum.A range from 21 to 27 ℃was fit for the cultivation,but the growth rate and final population density at 27 ℃would be higher than those at other temperatures. Practically,an optimum inoculating density should be set between 10×10~4 and 30×10~4/mL Though hardly any difference has been shown between total harvest amounts of batch and semicontinuous cultivation,the latter is still better for simplifying manipulation and reducing workload.国家自然科学基金(30470938);; 福建省自然科学基金(D0510002);; 厦门市科技计划项

THE LABORATORY CULTURE AND REPRODUCTION of TWO IANCELETS IN XIAMEN

国家自然科学基金(30570208);教育部高等学校博士学科点专项科研基金(20070384041)资