金鱼hAT家族转座子Tgf2的克隆及其结构

hAT家族转座子以果蝇hobo、玉米Ac和金鱼草(Ceratophyllum demersum L.)Tam3为代表,以"剪切-粘帖"方式进行DNA转座。1996年,日本学者首次在白化青鳉(Oryzias latipes)中发现具有天然活性的脊椎动物hAT家族转座子,即青鳉Tol2转座子,该转座子已在模式生物斑马鱼转基因、基因和启动子捕获方面进行了广泛应用。文章根据玉米Ac与青鳉Tol2转座子序列保守区设计一对引物,在19种不同鱼类物种或品系中进行PCR筛选,最后发现此类hAT家族转座子在我国不同品系金鱼中存在,命名为金鱼Tgf2转座子。金鱼Tgf2转座子全长4720bp,由4个阅读框组成,与青鳉Tol2转座子的相似度为97%。金鱼Tgf2与青鳉Tol2转座子在末端倒位重复和亚末端重复上存在一定差异,此外,金鱼Tgf2转座子的中间反向重复序列(1453bp到2091bp)可形成一种"十"字结构,明显有别于青鳉Tol2转座子形成的茎环结构,这些区域与转座活性密切相关。文章预示金鱼Tgf2转座子可能具有更高的天然转座活性,构建高效金鱼Tgf2转基因元件可供鱼类转基因和基因捕获研究。     

Tgf2转座子多克隆位点的克隆与表达

以金鱼pTgf2-EF1α-EGFP转座子为基础,构建含有多克隆位点(Multiple cloning sites,MCS)序列以及外源目的基因肌醇-3-磷酸合成酶(Myo-inositol-3-phosphate synthase,MIPS)的重组表达载体并注射到斑马鱼1-2期受精卵,检测重组表达载体pTgf2-EF1α-MCS-EGFP和pTgf2-EF1α-MCS-MIPS-EGFP在斑马鱼中绿色荧光蛋白基因(EGFP)的表达情况以及外源基因MIPS在斑马鱼体内的整合情况。荧光观察结果显示,两个重组载体均不影响EGFP的表达,只是表达强度存在一定差异,表明对Tgf2转座子的改造是有效的。转基因斑马鱼PCR检测结果显示,靶基因MIPS的编码区能够完整地整合到斑马鱼基因组中,整合效率达31.4%。重组表达载体的成功构建显示金鱼Tgf2转座子可以介导外源基因在斑马鱼中的表达,为以后Tgf2转座子在鱼类基因功能研究方面奠定了基础。     

转座子插入宿主基因在稻属中的分布式样及其适应意义的初探

转座子在各类真核生物基因组中都占有很高的比例,它们对宿主基因组特别是关联的基因在结构、功能和进化上都起着重要的作用。基于生物信息学分析,本研究选择了水稻基因组中2个被转座子插入的宿主基因,通过PCR扩增和琼脂糖凝胶电泳分析,获得了转座子在稻属16个代表物种94份材料中的插入式样。结果表明,这2个转座子在稻属中的分布式样与插入时间不同,基因三DG-＆02926349中的转座子在AA-基因组的物种中全部存在,基因LOC-Os02945130中的转座子则插入稻属AA-基因组的部分物种中,与AA-基因组的物种的系统发育关系相吻合。转座子在宿主基因组中不同的分布与保留式样以及插入后已经固定在不同地理来源的群体中,暗示了它们在物种进化过程中对宿主基因可能存在适应性意义。     

转座子插入位点的鉴定方法

转座子是基因组中的可移动成分,已经成为分子生物学研究中的一种有用的研究工具。转座子插入位点识别的难易决定着转座子应用的潜能。本介绍曾应用过的几种转座子插入位点周围染色体序列的克隆测序方法及其优缺点。     

Mutator转座子及MULE在植物基因与基因组进化中的作用

Mutator（Mu）转座子是植物中已发现的转座最活跃的转座子,其高的转座频率及趋向于单拷贝功能基因转座的特性,使该转座子成为玉米功能基因克隆的主要方法.Mu转座子的同源类似因子广泛存在于被子植物基因组中,而且同一基因组中往往具有多种变异类型.它不仅具有其他DNA转座子在基因和基因组进化中的普遍作用,而且具有能够承载基因组内功能基因和基因片段的载体功能,这种载体Mu转座子（Pack-MuLEs）能够在基因组内移动众多的基因片段,从而对基因和基因组进化产生作用.Mu转座子的同源序列发生在水稻与狗尾草之间的水平转移提供了高等植物核基因水平转移的首个例证.对Mu转座子的了解促进了我们对动态基因组概念的认识.文章对Mutator转座子的发现、转座特征、基因标签应用等的研究进展进行了综述,对Mu转座子家族的同源序列进行了分类,讨论了该转座子在基因组进化中的作用,分析了应加强研究的问题.     

人类基因组中的反转录转座子

人类基因组中有35%以上的序列为转座子序列.反转录转座子是引起人类疾病的潜在病因.人类基因组中的主导转座子——L1反转录转座子内部有二个开放读框,其编码蛋白具有RNA结合蛋白、反转录酶和内切酶活性.L1可能通过靶引物反转录机制整合到染色体中;Alu等非自主性反转录转座子可能利用L1反转录酶的反式互补作用进行转座.     

转座子挽救法对转座子突变菌株中插入位点的定位分析

为寻找谷氨酸棒杆菌转座子插入突变菌株中的转座子插入位点,采用了转座子挽救法对转座子及其插入位点附近的序列进行分离,并测定插入位点相邻DNA序列,获得了三个转座子插入位点DNA序列,其中一个是柠檬酸合成酶基因,另两个为目前未知基因,暂命名为orfA和orfB。该方法简便易行,是分析转座子插入位点的理想方法。     

高等植物基因组中的反转录转座子

高等植物基因组中的反转录转座子王石平张启发（华中农业大学作物遗传改良国家重点实验室武汉４３００７０）关键词植物,反转录转座子,遗传多样性,转座ＲＥＴＲＯＴＲＡＮＳＰＯＳＯＮＳＩＮＴＨＥＧＥＮＯＭＥＳＯＦＨＩＧＨＥＲＰＬＡＮＴＳＷＡＮＧＳｈｉＰｉｎ...     

Tol2转座子系统在转基因动物中的应用

Tol2是在青鳉鱼基因组中发现的一种具有自主性的转座子元件.它编码转座酶,催化Tol2转座子结构中5’端200 bp和3’端150 bp序列发生转座反应.Tol2的多种特性,如可携带大片段外源DNA、单拷贝整合效率高、转座子活性强等,使得以Tol2特座子系统为载体的转基因技术在多种生物中得到应用.综述了Tol2转座子系统的结构、特性以及近年来在多种动物转基因中的应用.     

插入突变在水稻功能基因组学中的研究进展

构建饱和的基因突变体库是最直接、有效的分析鉴定基因功能的方法.根据插入突变源不同可分为T-DNA插入突变、转座子插入突变等.主要介绍这两种方法的原理及其在水稻功能基因组学研究中的应用和进展,并分析和讨论了插入突变在水稻功能基因组学研究中存在的困难和发展趋势.     

Molecular cloning and function analysis of insulin-like growth factorbinding protein 1a in blunt snout bream(Megalobrama amblycephala)

Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream(Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.     

团头鲂黏蛋白基因Muc5b克隆及表达分析

摘要：黏液（mucus）在鱼体防御外界病原侵袭、信息传递、调节渗透压等方面具有重要作用。黏蛋白（mucin）作为黏液的基础骨架组分,与其相关的研究正受到广泛的关注。在本研究中,作者克隆获得团头鲂（Megalobrama amblycephala）Muc5b mRNA 的部分序列3895 bp,并通过qRT-PCR分析了Muc5b在团头鲂不同组织的表达分布及其在捕捞应激后在鳃和表皮中的表达变化。序列分析结果显示,团头鲂Muc5b与鲤等脊椎动物的Muc5b有较高的同源性,其N端含有黏液蛋白特异性结构域：三个VWD区域,三个C8区域,二个TIL区。组织表达分析结果表明,Muc5b在鳃和表皮表达量相对较高,在脑、脾、肾中表达水平较低,在肝、肠道几乎不表达。捕捞应激后1 h时鳃中Muc5b显著降低（P < 0.05）,24 h时恢复初始水平;表皮中4 h时Muc5b显著上升（P < 0.05）,24 h时恢复到初始水平。     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

水流对团头鲂(Megalobrama amblycephala)幼鱼游泳行为的影响

为探讨团头鲂幼鱼(Megalobrama amblycephala)游泳行为对水流的响应规律,该文通过特制鱼类游泳行为测定装置,测定了团头鲂幼鱼在25℃,0、0.1、0.2、0.3、0.4m/s流速条件下的游速、游距、转角、至中心点的距离及游泳轨迹。结果表明:随着流速的增大,个体游速、游距及转角值均相应增大。0、0.1及0.2m/s流速组间的游速、游距及转角差异均不显著(P>0.05),但显著小于0.3和0.4m/s组别,且0.3和0.4m/s流速组之间差异均不显著(P>0.05);整个时间段内,个体至中心点的距离随流速增大并未呈现明显规律性,各流速间差异不显著(P>0.05),游速与游距呈显著线性正相关,而与转角呈显著线性负相关,与至中心点的距离则无相关性;游泳轨迹随水流增大趋向复杂化。     

Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori Strains

The transformation rate of three different strains of silkworm Bombyx mori was comparedafter the introduction of enhanced green fluorescence protein (EGFP)-encoding genes into the silkwormeggs by microinjection of a mixture of piggyBac vector and helper plasmid containing a transposase-encodingsequence.Although there were no significant differences among the three strains in the percentages offertile moths in microinjected eggs (P=0.1258),the percentages of G_0 transformed moths in fertile mothsand injected eggs were both significantly different (P=0.01368 and P=0.02398, respectively).Thetransformation rate of the Nistari strain (Indian strain) was significantly higher than that of the other twostrains,Golden-yellow-cocoon (Vietnamese strain) and Jiaqiu (Chinese strain),which had similar rate. Theseresults indicate that the transformation efficiency of the piggyBac-based system might vary with silkwormstrains with different genetic backgrounds.The presence of endogenous piggyBac-like elements might bean important factor influencing the transformation efficiency of introduced piggyBac-derived vectors,andthe diverse amount and activation in different silkworm strains might account for the significant differences.     

Identification of a novel helitron transposon in the genome of Antarctic fish

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9 kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated.     

水稻Ds插入纯合体的筛选和鉴定

采用Basta抗性鉴定、潮霉素抗性鉴定和PCR检测相结合的方法筛选和鉴定了水稻Ds插入纯合体。在T1代236个转化株系中,有16个株系的全部植株表现出对Basta的敏感,其余220个株系的植株表现出对Basta的抗性。经过3代的纯合筛选,共鉴定出Ds插入纯合体203个．这些Ds插入纯合体可用于构建Ac／Ds系统和对Ds插入突变体进行筛选和鉴定,为水稻功能基因组学研究提供了材料。     

Insertion of a transposon for chloramphenicol resistance into bacteriophage Mu.

We have isolated mutants of bacteriophage Mu carrying the X mutations caused by the insertion of cam (Tn9), a transposon for chloramphenicol resistance. The Mu X cam mutants were obtained by selecting for heat-resistant survivors of a Mucts62, P1cam dilysogen. Like the previously described X mutants, Mu X cam mutants are defective prophages which can be excised from the host DNA at a frequency of 10(-5) to 10(-7) per cell. Tn9 insertions in Mu X cam mutants are located within 5000 base pairs of the left end of Mu DNA in a region that controls early replication functions of Mu. There is one EcoRI cleavage site in Tn9. The Tn9 transposon itself can be excised precisely from the Mu X cam mutants to generate wild type Mu. In most Mu X cam mutants, precise excision of Tn9 occurs at a low frequency (10(-6) per cell), whereas in some, the frequency is higher (10(-4) per cell). Mu X cam prophages can replicate after induction with the help of wild type Mu. The lysates containing Mu X cam particles, however, fail to transduce chloramphenicol resistance at a high frequency; Mu X cam mutants apparently have a cis dominant defect in integration.     

Isolation and characterization of polymorphic microsatellite loci in Wuchang bream (Megalobrama amblycephala)

Wuchang bream (Megalobrama amblycephala) is an economically important fish in China. From a (GT)₁₃‐enriched genomic library, 20 microsatellites were developed. Nine of these 20 loci were polymorphic in a test population with allele numbers ranging from two to four, and the observed and expected heterozygosities ranging from 0.2609 to 0.7826 and from 0.3739 to 0.7546, respectively. In the cross‐species amplifications, six of these nine loci were also polymorphic in white amur bream (Parabramis pekinensis). These polymorphic microsatellite loci are potentially useful for population genetics of Wuchang bream and its closely related species.