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

SSR Polymorphism of Alligator sinesis and Conservation Strategy of Genetic Diversity

作者简介: 黄 磊( 1978- ) , 硕士研究生, 研究方向: 动物分子遗传 通讯作者。E-mail:wangyqnj@ jlonline. com[中文文摘]扬子鳄 (Alligatorsinensis)是中国特有的珍稀保护动物 ,为保护这一濒危物种 ,我国于 80年代初在安徽宣州先后建立了扬子鳄繁殖研究中心和国家级扬子鳄自然保护区 ,现饲养种群存鳄数量已达 10 0 0 0余头。为了揭示扬子鳄种群的遗传结构 ,共采集了 3 9个个体的样品 ,其中包括 6件剥制标本 ,按代系不同 ,分为野生群、F1代饲养群及F2 代饲养群 ,用微卫星DNA分子标记对其进行研究。分析结果显示 :扬子鳄种群在微卫星水平表现出很低的遗传多样性 ,平均等位基因数A =2 3 8,平均有效等位基因数Ne=1 60 ,平均观察杂合度Ho=0 3 74,平均期望杂合度He=0 3 50 ,平均多态信息含量PIC =0 3 2 7,3个群体间A、Ne、Ho、He、PIC及各微卫星位点等位基因频率分布无显著差异 ,但F2 代饲养群在Ami μ 6和Ami μ 2 2 2两个位点表现出极显著的遗传不平衡。扬子鳄种群遗传多样性水平低下的主要原因是近几十年来种群数量大幅减少造成 ,现阶段应将全部现存的扬子鳄作为一个整体加以保护 ,在建立新的繁殖群体时 ,应考虑保存物种遗传多样性所必需的有效种群大小 ,在种群的遗传管理上应注重低频等位基因的发现和保持.[英文文摘]Chinese alligator, Alligator sinesis , is a critically endangered endemic species under legislative protection. Results of recent investigations revealed that the number of the alligator was continuously declining in the past 50 years and less than 150 individuals were surviving in the wild until 2000. In order to prevent the extinguishing of this species, the Reproductive Research Center of Alligator sinesis and the National Nature Reserve of Alligator sinesis were set up in early 1980 s in Xuanzhou, Anhui Province. After 20 years of breeding efforts, the number of captive individuals has been brought up to more than 10, 000 in total. In order to reveal the genetic structure of Chinese alligator population, total of 39 individuals including 7 wild individuals outside of the research center were sampled to construct wild, F1 and F2 groups according to their generations, and 10 micorsatellite loci selected from 25 primer pairs originally designed for Alligator mississippiensis were employed for investigating the genetic diversity of Alligator sinesis .The results indicated that, contrasting with Alligator mississippiensis and some other endangered species, Chinese alligator had an extremely low genetic diversity level with A= 2.38, Ne= 1.60, Ho= 0.374, He= 0.350 and PIC= 0.327. There were no significant differences of A, Ne , Ho , He , PIC and each SSR locus alleles frequency distribution among 3 groups. However, Hardy Weinberg equilibrium analysis revealed that F2 captive group showed a remarkable genetic disequilibrium at loci Ami μ 6 and Ami μ 6 222 . The reason accounting for the current genetic status of Chinese alligator is dramatically shrink of the population in past decades. Due to the lack of significant difference between wild group and captive group, all survived Chinese alligator should be treated as one ESU in the next conservation practice. More attention regarding the effective population size and low frequency alleles should be emphasized in genetic management of captive alligators and establishing new separate propagation.教育部骨干教师资助计划项目(编号:GG18021002403 1740);教育部留学回国人员启动基金资

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~

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

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

Progress in the study of Pax gene family and its alternative splicing

Pax基因家族编码的蛋白是一组极为重要的转录调控因子,在胚胎发育的器官形成中扮演重要角色,其主要功能包括:调控细胞增殖、促进细胞自我更新、诱导前体细胞定向转移以及改变特异细胞系的分化方向。目前已知,Pax基因的非正常表达是多种先天性疾病的主要诱因。Pax基因的选择性剪接体通常具有一定的空间特异性,每种剪接体都有其主要作用的靶位和信号通路。文章简述了Pax基因的相关背景知识,详细介绍Pax1—Pax9调控在胚胎组织发育中的各项功能,并列举了现已确定的Pax基因在不同物种中的选择性剪接产物。The Pax gene family encodes a group of transcription factors and plays an essential role in organogenesis during embryonic development through regulating cell proliferation and self-renewal, migration of embryonic precursor cells, and the coordination of specific differentiation programs. This is supported by the fact that expression of Pax is dysregulated in several congenital disease. Alternative transcripts of Pax genes are identified in various tissues with specific location, and each isoforms has their own major molecular targets and signaling pathways. In this review, we provide a general background of the Pax genes, highlight the role of Pax proteins playing within specific tissues in terms of embryological development, and give a brief introduction of alternatively spliced isoforms of Pax protein identified.国家自然科学基金(30470938,30570208

The application of microsatellite DNA markers in conservation genetics of endangered animals

通讯作者Author for correspondence. E-mail : wangyq @xmu. edu. cn[中文摘要]微卫星DNA广泛分布于真核生物基因组中 ,具有多态性高、共显性遗传、选择中性、易于操作等特点 ,是一种极具应用价值的分子遗传标记 ,近年来在濒危动物保护遗传学研究中得到越来越多的应用。微卫星DNA高度多态性提供的高分辨率遗传信息 ,使其不仅适合个体水平的亲子鉴定与交配系统研究 ,而且也已成为种群遗传结构与多样性分析的有效分子标记。微卫星分析所需的DNA量极少 ,用非损伤性方法获取的极少量样品或陈旧样品就能用于有效分析 ,方便了濒危动物野外调查工作的开展 ,并且可以利用年代久远的馆藏历史标本揭示种群的重要历史进程。另外 ,某些微卫星DNA大小在近缘物种间可相互区分 ,这使得部分物种的DNA分子鉴别将更为简便。但微卫星分子标记的座位筛选和特异引物开发耗时费力 ,一定程度上限制了其广泛应用。针对不同的研究目的选择合适的分子标记方法将有助于更好的揭示问题本质.[英文摘要]Microsatellite DNA is widely dispersed in eukaryotic genomes with the characters of high polymorphism , high abundance , codominance , selective neutrality , and easy manipulation. Therefore , it has been increasingly applied to studies of conservation genetics of endangered animals in recent years. The polymorphism of microsatellite DNA is so high that it can provide excellent resolution not only for kinship and mating system studies at the individual level , but also for genetic structure research at the population level . The DNA template needed for microsatellite analysis is very low and has no special demands , so that small samples obtained with noninvasive method and from old specimen can be analyzed effectively. Therefore , the approach not only makes investigation of endangered animals surviving in the wild more convenient and exact , but also can make use of the rare specimens preserved in museums to reveal important evolutionary history for some species. Fur2 thermore , some microsatellite fragments′sizes can be discriminated among related species , which makes it possible to identify species more conveniently with only fragment analysis. However , application of microsatellite DNA markers also has its own shortcomings , and appropriate molecular makers should be adopted for a given issue according to different research purposes.教育部留学回国人员启动基金;教育部高等学校骨干教师资助计划项目 (GG 180 2 10 0 2 40 3 1740

Construction of subtractive cDNA library of the gonad of amphioxus Branchiostoma japonicum

本研究以日本文昌鱼(Branchiostoma japonicum)性腺cDNA为材料,应用抑制性差减杂交技术构建文昌鱼雌雄性腺的差减cDNA文库。正向差减杂交以卵巢为试验方、精巢为驱动方,反向差减杂交以精巢为试验方、卵巢为驱动方,将所获差减cDNA片段克隆插入质粒表达载体,转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含459、243个重组子。PCR扩增鉴定正、反向差减cDNA文库的插入片段,其中90%左右的克隆皆能扩增出有效产物,插入片段范围为200-600bp,符合差减文库PCR产物片段的大小。随机选取30个阳性克隆测序分析,得到26有效基因片段,进一步从中选取16个序列,用实时定量PCR对文库质量进行验证,结果表明所建文库能够达到富集雌雄性腺差异表达基因的目的。文昌鱼性腺差减文库的构建,为进一步分离、鉴定性腺分化和发育相关基因奠定了基础。In the present study,two subtracted cDNA libraries for gonads of amphioxus were constructed using the suppression subtractive hybridization(SSH) technique.cDNA from ovary was adopted as tester for the construction of a forward library,and then as a driver for the reverse library construction.Two-directional subtracted cDNA fragments were inserted into plasmid vectors,and subsequently the vectors were transferred into E.coli DH5α.As a result,forward and backward subtracted cDNA libraries containing 459 and 243 clones respectively,were obtained and subjected to PCR analysis.Results confirmed that about 90% of the clones contained inserts of 200-600 bp in the two libraries,in accord with our prediction.Thirty of the positive clones were selected and sequenced randomly,and,of those sequenced clones,twenty-six gene fragments were obtained.Then,sixteen sequenced genes were further analyzed via Real-time PCR method.The results indicated that these genes were expressed differently between male and female gonads.The subtractive cDNA libraries of amphioxus gonads provide a foundation for further studies of the genes related to sexual differentiation and gonadal development.国家自然科学基金资助项目(No.30470938,No.30570208)~

Establishment of full-sib families of Branchiostoma japonicum and the relationship between early development patterns and larvae survival rates

2011年3月9日—5月19日,以厦门海域采集的野生个体和实验室繁育的日本文昌鱼成体为亲本,尝试性地建立了全同胞家系;采用温度和光照诱导的方法,对雌、雄亲本进行人工催产,获得了34对亲本的全同胞家系受精卵。在家系培育过程中发现致使胚胎和仔鱼死亡的几种主要原因,以及日本文昌鱼早期发育的两种模式。通过加强养护管理,提高仔鱼存活率,缩短幼体变态所需时间,最终建立了7个完成变态的全同胞家系。在这些家系中,从受精卵到完成变态后不久的亚成体存活率最高为32.4%,最低为1.67%,而变态最快历时24 d,最慢历时42 d。虽然在全同胞家系建立过程中,幼体死亡率高,家系间的胚胎和幼体生长发育状况差异大,但实验结果表明日本文昌鱼全同胞家系建立完全可行,为其优良品系选育建立了基础。One general requirement of individual laboratory animals is that they have known genetic backgrounds.However, ensuring such genetic similarity is difficult, and can be facilitated by breeding a full strain for experimentation.To this end, the authors bred 34full-sib families of amphioxus larvae/embryos.Due to the high mortality of the embryos and larvae, only seven full-sib families of juvenile amphioxus Branchiostoma japonicum were obtained.Among them, the highest and lowest survival ratios were 32.4% and1.67%, respectively, whereas the shortest metamorphosis and longest larva duration were 24 d and 42 d, respectively.These results demonstrate the feasibility of establishing full-sib families of amphioxus, and provide fundamental data needed for the future breeding of amphioxus strains.国家高技术研究发展计划(“863”)项目(2008AA092602); 国家自然科学基金项目(30830023); 深圳市科技研发资金项目(JSF201006290026A

Taxonomic and Molecular Phylogenetic Studies of Amphioxus: A Review and Prospective Evaluation

文昌鱼是最接近脊椎动物直接祖先的现生动物,在脊椎动物起源与演化研究中占有极其重要的位置。近年来,对文昌鱼的研究已引起越来越多的科学家的兴趣,然而作为生命科学研究的重要基础,这类动物的分类学研究相对滞后。依据已有的中国文昌鱼资源调查资料,中国沿海文昌鱼的分布应当十分广泛,即只要有适合文昌鱼栖息的沙滩,均有文昌鱼分布的可能。根据目前的分类学研究成果和动物命名法中的优先权原则,建议将产于青岛等地的文昌鱼种名Brnachiostoma belcheri tsingtauense订正为B.japonicus,南方的文昌鱼保留其原种名B.belcheri。由此,目前分布在中国沿海的鳃口文昌鱼属(Branchiostoma)至少有2种,侧殖文昌鱼属(Epigonichthys)有1～3种,漂浮文昌鱼(Amphioxides pelagicus)1种。DNA分子标记技术在文昌鱼分类学研究中将会发挥更大的作用。Amphioxus, as the closest living animal of the last common ancestor of all vertebrates, occupies an extremely important phylogenetic position in the evolution of vertebrates and has attracted the interest of scientists from various research fields in recent years. However, despite their importance for the life sciences, taxonomic studies of amphioxus are relatively limited. In present review, we summarize current progress in both field investigations and taxonomic research of Chinese amphioxus. Based on the investigation data, we assume that amphioxus is distributed in all habitable sandy beaches along the Chinese coast from south to north. According to the rule of priority and recent taxonomic studies on amphioxus, we also propose that the original subspecies Brnachiostoma belcheri tsingtauense together with the population in most Japanese waters is an independent species and its name should be revised to B. japonicus. Consequently, there are at least two species of genus Brnachiostoma and 1-3 of Epigonichthys and one of Amphioxides in the China Sea. Finally, the application of DNA molecular markers in systematic studies of cephalochordate is evaluated.国家自然科学基金资助项目(30470938;30570208);; 福建省自然科学基金资助项目(D0510002);; 厦门市科技计划项目(3502Z20042015