植物基因组中的反转录转座子

植物反转录转座子是植物基因组中一类可移动的遗传元件，可分为LTR反转录转座子和非LTR反转录转座子两类。反转录转座子以高拷贝广泛分布于植物基因组中，具有高度的异质性，可被某些生物或非生物逆境激活并通过纵向和横向在物种内和物种间传递，对植物基因组的结构、功能和进化起着重要的作用。文中对反转录转座子的类型、结构与特点进行综述，并探讨其作为分子标记在现代生物学中的应用。     

反转录转座子分子标记及其在植物研究中的应用

反转录转座子以高拷贝在植物基因组中广泛分布,具有高度的异质性,可作为分子标记对植物基因组进行研究.文章对SSAP、IRAP、REMAP及RBIP4种基于植物反转录转座子开发的分子标记技术的原理、技术流程、特点及其在植物研究中的应用进行了概述.     

Tn7转座子在大肠杆菌、迟钝爱德华氏菌及鳗弧菌中的应用

Tn7转座子是一种定点插入型转座子,固定地插在细菌的glmS基因下游的attTn7位点,而glmS基因与细胞壁的合成有关,存在于所有的细菌中,所以Tn7转座子可以插入到所有细菌的染色体,因此Tn7转座系统已被开发为细菌基因组改造的重要工具。为了方便对大肠杆菌、迟钝爱德华氏菌和鳗弧菌的改造和研究,探讨应用Tn7转座子对其基因组进行改造的操作步骤,并利用阿拉伯糖启动子pBAD诱导的Flp/FRT系统建立了高效、快捷的抗性消除方法。     

亚洲棉全基因组中NAC类转录因子基因的鉴定与分析

为了开发棉花NAC基因资源,本文利用生物信息学方法在亚洲棉基因组中预测了138个NAC基因,根据其在染色体上的位置关系命名为Ga NAC001-Ga NAC138,所有Ga NAC基因分布于亚洲棉13条染色体上。根据Ga NAC蛋白的序列相似性及系统进化分析,将亚洲棉Ga NAC蛋白分为11个亚家族,每个亚家族的Ga NAC蛋白数目为4-25个。基因结构分析发现,大部分(63.7%)Ga NAC基因含有2个内含子。比较亚洲棉与陆地棉、雷蒙德氏棉NAC蛋白的同源性,发现亚洲棉与雷蒙德氏棉NAC蛋白的匹配程度高于陆地棉,平均序列一致性分别为93.9%和81.2%,一些NAC基因(58/138)在棉属的进化过程中高度保守,一些(37/138)则变异程度较大,可能是生物体为了适应环境的变化产生了新的功能。本研究结果为亚洲棉及陆地棉NAC转录因子基因的后续深入研究提供了重要参考。     

哺乳动物的反转录转座子及其在进化中的作用

哺乳动物基因组中整合了大量反转录转座子。根据它们的结构特点和起源，对反转录转座子的分类、扩增途径和扩增酶学进行了探讨，提出了反转录转座子本身的进化方向及其在基因组进化中的作用。依据原祖７ＳＬＲＮＡ演化为人类Ａｌｕ家族的过程，讨论了Ａｌｕ序列在基因表达调节中的作用。     

人气管普孢子虫基因组中MITEs转座子的鉴定及进化分析

利用生物信息学方法,首次在人气管普孢子虫(Trachipleistophora hominis)基因组内鉴定到9个MITEs家族ThME1~ThME9,共123个拷贝,MITEs转座子的长度均小于600bp。根据靶位点重复序列(Target site duplication,TSD)的不同,将ThME1归属于Tc1/Mariner超家族,ThME2和ThME3归属于PIF/Harbinger超家族,ThME5和ThME6归属于CACTA超家族(超家族的名字用正体较好,下同),其余家族归为新家族。分析发现,人气管普孢子虫中的所有MITEs家族的吉布斯自由能均小于0,表明MITEs家族具有形成二级结构的潜能,有利于该家族在基因组上转座。人气管普孢子虫MITEs家族在基因组的插入时间估计在0~800万年内,而且这种插入在基因组中是随机的,没有位置偏向性,并发现2个MITEs转座子拷贝插入到基因编码区内部。上述结果为进一步研究微孢子虫MITEs转座子的起源以及功能奠定了基础。     

PiggyBac 转座子在肝脏中的活性研究

本实验运用高压尾静脉注射的方法将PB转座系统导入小鼠肝脏,研究其在肝脏中的转座活性,系统中PB转座子携带表达红色荧光蛋白的基因以指示转座的发生.注射10个月后取出小鼠肝脏,体视荧光显微镜观察肝脏是否有红色荧光,并通过基因组PCR、splinkerette PCR分析PB转座子在肝脏中的插入情况.实验结果表明,PB转座子在肝脏内发生高效转座,检测的68个PB插入位点中有34个位于基因序列,使得PB系统可以作为有效的基因诱变工具来研究基因功能.     

柞蚕微孢子虫基因组LTR反转座子鉴定与进化分析

【目的】鉴定柞蚕微孢子虫(Nosema pernyi)基因组中LTR反转座子的种类、数量和遗传关系。【方法】从GenBank数据库下载柞蚕微孢子虫基因组序列,采用MGEScan软件筛选潜在重复元件,通过NCBI-BLAST与不同物种的逆转酶进行比对,含有逆转录酶结构域的序列认定为LTR反转座子,并探讨它们的结构特征、所属类型和基因组分布特点。【结果】从柞蚕微孢子虫基因组中共发现6个LTR反转座子家族即Nar1,Nar2,Nar3,Nar4,Nar5和Nar6,长度在3～4kb之间;大部分家族有相似的LTR末端核苷酸,但靶位点重复和拷贝数存在差异;逆转录酶结构域系统进化树显示6个LTR反转座子家族均属于Ty3/Gypsy类型,且与家蚕微孢子虫(Nosema bombycis)LTR反转座子形成姊妹分支;Nar2和Nar4家族在基因组上成串排列,具有相似的分布特征。【结论】柞蚕微孢子虫基因组中存在Ty3/Gypsy类型的LTR反转座元件,进化地位与家蚕微孢子虫最为接近;LTR反转座子家族重复元件在基因组上成串排列,可能是柞蚕微孢子虫基因组扩张的潜在因素之一。     

人气管普孢子虫基因组中MITEs转座子的鉴定及进化分析

利用生物信息学方法,首次在人气管普孢子虫( Trachipleistophorahominis )基因组内鉴定到 9 个 MITEs 家族ThME1~ThME9 ,共 123 个拷贝, MITEs 转座子的长度均小于 600bp 。根据靶位点重复序列( Targetsiteduplication ,TSD )的不同,将 ThME1 归 属 于 Tc1 / Mariner 超 家 族, ThME2 和 ThME3 归 属 于 PIF / Harbinger 超 家 族, ThME5 和ThME6 归属于 CACTA 超家族(超家族的名字用正体较好,下同),其余家族归为新家族。分析发现,人气管普孢子虫中的所有 MITEs 家族的吉布斯自由能均小于 0 ,表明 MITEs 家族具有形成二级结构的潜能,有利于该家族在基因组上转座。人气管普孢子虫 MITEs 家族在基因组的插入时间估计在 0~800 万年内,而且这种插入在基因组中是随机的,没有位置偏向性,并发现 2 个 MITEs 转座子拷贝插入到基因编码区内部。上述结果为进一步研究微孢子虫 MITEs 转座子的起源以及功能奠定了基础。
     

茄子反转录转座子基因片段的克隆及序列分析

根据己报道的反转录转座子的序列设计合成一对特异引物,以西安绿茄基因组DNA为模板,采用PCR扩增的方法扩出一条DNA特异片段并克隆到pMD18-T载体中.用PCR法和酶切分析法对克隆片段进行鉴定并进一步进行核苷酸序列分析.序列测定该片段长为518 bp.用GenBank中的BLAST程序分析表明,该片段与马铃薯(Solanum demissum)反转录转座子的gag-pol聚合蛋白90-243aa区域氨基酸同源性为43%.     

Primary investigation on GISH-NOR in cotton

Six loci of nucleolar organizer region (NOR) were detected in genomic in situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted from Gossypium species as probe. With twice FISH to the same mitotic cell of G herbaceum or G hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs from G hirsutum and Graimondii mitotic images were all terminal types. Four and two GISH-NORs from G herbaceum (var. africanum) were terminal and centromere types, respectively. Six GISH-NORs in G hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image of G raimondii with its own gDNA as probe. From mitotic image of G herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-wholerecovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes in Gherbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.     

Host genome surveillance for retrotransposons by transposon-derived proteins

Transposable elements and their remnants constitute a substantial fraction of eukaryotic genomes. Host genomes have evolved defence mechanisms, including chromatin modifications and RNA interference, to regulate transposable elements. Here we describe a genome surveillance mechanism for retrotransposons by transposase-derived centromeric protein CENP-B homologues of the fission yeast Schizosaccharomyces pombe. CENP-B homologues of S. pombe localize at and recruit histone deacetylases to silence Tf2 retrotransposons. CENP-Bs also repress solo long terminal repeats (LTRs) and LTR-associated genes. Tf2 elements are clustered into 'Tf' bodies, the organization of which depends on CENP-Bs that display discrete nuclear structures. Furthermore, CENP-Bs prevent an 'extinct' Tf1 retrotransposon from re-entering the host genome by blocking its recombination with extant Tf2, and silence and immobilize a Tf1 integrant that becomes sequestered into Tf bodies. Our results reveal a probable ancient retrotransposon surveillance pathway important for host genome integrity, and highlight potential conflicts between DNA transposons and retrotransposons, major transposable elements believed to have greatly moulded the evolution of genomes.     

Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana

The genome of the flowering plant Arabidopsis thaliana has five chromosomes. Here we report the sequence of the largest, chromosome 1, in two contigs of around 14.2 and 14.6 megabases. The contigs extend from the telomeres to the centromeric borders, regions rich in transposons, retrotransposons and repetitive elements such as the 180-base-pair repeat. The chromosome represents 25% of the genome and contains about 6,850 open reading frames, 236 transfer RNAs (tRNAs) and 12 small nuclear RNAs. There are two clusters of tRNA genes at different places on the chromosome. One consists of 27 tRNA(Pro) genes and the other contains 27 tandem repeats of tRNA(Tyr)-tRNA(Tyr)-tRNA(Ser) genes. Chromosome 1 contains about 300 gene families with clustered duplications. There are also many repeat elements, representing 8% of the sequence.     

Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L

Mammalian genomes employ heritable cytosine methylation in the long-term silencing of retrotransposons and genes subject to genomic imprinting and X chromosome inactivation. Little is known of the mechanisms that direct cytosine methylation to specific sequences. Here we show that DNA methyltransferase 3-like (Dnmt3L (ref. 1)) is expressed in testes during a brief perinatal period in the non-dividing precursors of spermatogonial stem cells at a stage where retrotransposons undergo de novo methylation. Deletion of the Dnmt3L gene prevented the de novo methylation of both long-terminal-repeat (LTR) and non-LTR retrotransposons, which were transcribed at high levels in spermatogonia and spermatocytes. Loss of Dnmt3L from early germ cells also caused meiotic failure in spermatocytes, which do not express Dnmt3L. Whereas dispersed repeated sequences were demethylated in mutant germ cells, tandem repeats in pericentric regions were methylated normally. This result indicates that the Dnmt3L protein might have a function in the de novo methylation of dispersed repeated sequences in a premeiotic genome scanning process that occurs in male germ cells at about the time of birth.     

CENP-B preserves genome integrity at replication forks paused by retrotransposon LTR

Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats.