裸燕麦Ty1-copia类反转录转座子反转录酶序列的分离、鉴定与分析

本研究根据Ty1-copia类反转录转座子反转录酶的保守区设计简并引物,通过PCR扩增,从裸燕麦(Avena nuda L.)品种‘品燕1号’基因组中分离获得23条Ty1-copia类反转录转座子序列,并对序列特征、系统发育关系及其转录活性进行分析。结果显示,23条Ty1-copia类反转录转座子存在较高的异质性,序列间的一致性为45%～98%,存在插入、移码和终止密码突变,但频率不高;系统发育分析结果表明,燕麦Ty1-copia类反转录转座子在进化过程中主要为垂直传递。本研究通过检索燕麦基因表达数据库,发现了5个有转录活性的Ty1-copia类反转录转座子。     

BB染色体组野生种花生LTR反转录转座子RT基因的多样性分析

利用简并PCR技术从野生花生种(Arachis ipaensis Krapov.et W.C.Greg.)的基因组中扩增分离Ty1-copia类(1类)和Ty3-gypsy类(2类)反转录转座子RT基因,并对其序列特征、多样性、系统进化关系及转录活性进行分析.结果显示:对于1和2类RT基因,目的条带分别约为260和43...     

苹果Ty1-copia类逆转座子家族鉴定及特性分析

根据逆转座子RT保守序列设计引物,利用PCR方法从苹果'嘎拉'中克隆了20条RT片段,分析苹果基因组内Ty1-copia类逆转座子家族特性及进化关系.结果显示,20条逆转录酶保守序列表现出了高度的异质性.结合已报道的37条苹果Ty1-copia类逆转座子RT片段,构建了系统发育树,发现家族1、3和4中具有转座活性的逆转座子的可能性较大;序列分析表明,Ty1-copia类逆转座子是苹果基因组内序列重组的热点.用RT序列为探针进行Southern杂交,发现苹果基因组内Ty1-copia类逆转座子拷贝数高、分布广泛.研究结果为进一步分离具有转座活性的苹果Ty1-copia类逆转座子及其人工诱导芽变奠定了基础.     

绿豆Ty1/Copia类反转录转座子逆转录酶序列的克隆和分析

利用Ty1/copia类反转录转座子的保守位点设计简并引物,从绿豆(Vigna radiata(L.)Wilczek)基因组中扩增得到了反转录转座子的逆转录酶序列.对扩增得到的约270bp的片段进行分离和克隆,并随机挑选了40个克隆进行测序,结果得到了36个单独的核酸序列,其中18个含有移码突变或终止子.根据序列比对,这些克隆可分为9组以及单个的9种.这40个克隆中,核酸序列相似性从8%到100%,显示出其核酸序列的高度异质性.将这些克隆的核酸序列与来自其他种植物的相应序列进行谱系分析,发现有些克隆与来自其他种植物的相应序列的亲缘关系比这些克隆之间更为接近.斑点杂交显示Ty1/copia反转录转座子约占绿豆基因组的9.3%.     

植物反转录转座子及其在功能基因组学中的应用

高等植物中的反转录转座子是构成植物基因组的重要成分之一.它分病毒家族和非病毒家族两类,病毒家族包括反转录病毒和类似于反转录病毒的非病毒转座子,病毒家族中的反转录转座子可再细分为Ty3-gypsy类和Ty1-copia类;非病毒家族可细分为LINE类和SINE类.正常情况下大部分反转录转座子不具有活性,某些生物或非生物因素胁迫可激活部分反转录转座子转座.反转录转座子自身编码反转录酶进行转录,以"拷贝-粘贴"的转座模式导致基因组扩增和进化.具有活性的反转录转座子通过插入产生新的突变,可作为一种基因标签技术,应用于功能基因组学研究,并成为研究植物基因功能和表达的重要技术平台.本文综述了近几年来在植物反转录转座子方面的研究进展,主要包括植物反转录转座子的结构、特征、活性及其对基因组的影响和它们在功能基因组学中的应用.     

绿豆Ty1／Copia类反转录转座子逆转录酶序列的克隆和分析

利用Ty1/copia类反转录转座子的保守位点设计简并引物,从绿豆(Vignaradiata(L.)Wilczek)基因组中扩增得到了反转录转座子的逆转录酶序列。对扩增得到的约270bp的片段进行分离和克隆,并随机挑选了40个克隆进行测序,结果得到了36个单独的核酸序列,其中18个含有移码突变或终止子。根据序列比对,这些克隆可分为9组以及单个的9种。这40个克隆中,核酸序列相似性从8%到100%,显示出其核酸序列的高度异质性。将这些克隆的核酸序列与来自其他种植物的相应序列进行谱系分析,发现有些克隆与来自其他种植物的相应序列的亲缘关系比这些克隆之间更为接近。斑点杂交显示Ty1/copia反转录转座子约占绿豆基因组的9.3%。     

节瓜Ty1-copia类逆转座子逆转录酶序列的克隆及比对

对8个节瓜（Benincasa hispida var.chieh-qua How）品系基因组DNA中的Ty1-copia类逆转座子逆转录酶核苷酸序列进行扩增,并对品系A39FA的29个克隆产物的核苷酸序列及翻译的氨基酸序列的系统进化和同源性进行了分析,还对29条氨基酸序列进行了比对。扩增结果表明：8个节瓜品系的基因组DNA中均包含长度约260 bp的逆转录酶核苷酸片段;从品系A39FA中获得的29条Ty1-copia类逆转座子逆转录酶核苷酸序列（CqRt1至CqRt29）的长度为247~267 bp,同源率为46.2%~98.1%,而它们的氨基酸序列同源率为26.7%~98.8%。序列分析结果表明：节瓜Ty1-copia类逆转座子逆转录酶核苷酸序列中碱基A、T、G和C的数量分别为65~96、47~92、45~74和32~49,所有序列均富含碱基A和T,AT/GC比为1.35~2.33;缺失突变是造成节瓜Ty1-copia类逆转座子逆转录酶核苷酸序列长度差异的主要因素,在序列长度和碱基组成方面的明显差异表明节瓜Ty1-copia类逆转座子逆转录酶核苷酸序列具有高度异质性。翻译后的氨基酸序列中有21条序列存在终止密码子突变、12条序列存在移框突变,表明Ty1-copia类逆转座子是节瓜基因组内序列重组的热点。通过聚类分析可将29个逆转录酶核苷酸序列分为5个家族（Family）,分别包括16、4、4、4和1条序列,其中Family 1可能是具有转座活性的逆转座子家族,但存在转录活性的逆转录酶序列仅占全部序列数量的20.69%。将每一家族中的1~2条序列与其他15种植物的Ty1-copia类逆转座子逆转录酶的氨基酸序列进行比对,显示出较高的同源性。研究结果表明：节瓜与其他植物的Ty1-copia类逆转座子可能有相同起源,而且Ty1-copia类逆转座子可在不同类群间横向传递。     

Copia 类型反转录转座子在籽粒苋中的表现

利用籽粒苋为材料 ,通过PCR扩增、克隆研究了copia类型反转录转座子的反转录酶序列在籽粒苋中的表现 ,结果表明 :(1)反转录转座子在苋属的 30个品系中同时检测到 ,说明反转录转座子在籽粒苋的不同种中普遍存在 ;(2 )对野生苋 (A .quitensis)中克隆、序列分析 2 8个反转录转座子的反转录酶序列 ,碱基分析表明 ,它们存在高度异质性 ,不同的反转录酶序列均存在不同程度的碱基缺失突变和终止密码子的突变 ;(3)对 2 8个序列与已发表的一些不同物种 (如水稻、矮牵牛和果蝇等 )的同一类型序列聚类分析表明 ,存在于野生苋中的反转录转座子与来源于双子叶或单子叶植物的反转录转座子关系密切 ,它们与果蝇的copia因子和 1731因子可能具有相同的起源     

不结球白菜Ty1-copia类逆转座子序列的克隆及表达

参照Ty1-copia类逆转座子逆转录酶的保守区设计简并引物,分别从10个不结球白菜(Brassica campestris ssp.chinensis)品种的全基因组中均扩增出260 bp左右的目标条带. 将目的条带回收、克隆和测序后进行分析,DNAstar分析发现,这些序列存在高度的异质性,28个核苷酸序列变化范围为224～278 bp,同源性范围为16.7%～83.0%.28条序列通过核苷酸聚类分为8个家族.推导氨基酸序列有移框突变、终止子突变或二者兼有;与已登录的不同物种同一类型逆转录酶氨基酸系统进化树分析表明,不结球白菜Ty1-copia类逆转座子与芥菜型油菜、拟南芥、芜菁、甜菜可能有共同的起源.半定量和实时定量PCR检测表明,水杨酸(salicylic acid)和Peronospora parasitica均能激活不结球白菜Ty1-copia类逆转座子,逆转座子在不结球白菜叶片中的表达特征说明它可能参与寄主对病原菌的抗性.     

Retrotransposon populations of <Emphasis Type="Italic">Vicia</Emphasis> species with varying genome size

The (non-LTR) LINE and Ty3-gypsy-type LTR retrotransposon populations of three Vicia species that differ in genome size (Vicia faba, Vicia melanops and Vicia sativa) have been characterised. In each species the LINE retrotransposons comprise a complex, very heterogeneous set of sequences, while the Ty3-gypsy elements are much more homogeneous. Copy numbers of all three retrotransposon groups (Ty1-copia, Ty3-gypsy and LINE) in these species have been estimated by random genomic sequencing and Southern hybridisation analysis. The Ty3-gypsy elements are extremely numerous in all species, accounting for 18–35% of their genomes. The Ty1-copia group elements are somewhat less abundant and LINE elements are present in still lower amounts. Collectively, 20–45% of the genomes of these three Vicia species are comprised of retrotransposons. These data show that the three retrotransposon groups have proliferated to different extents in members of the Vicia genus and high proliferation has been associated with homogenisation of the retrotransposon population.Electronic Supplementary Material Supplementary material is available for this article at .     

Presence ofenv-like sequences inQuercus suber retrotransposons

The main difference between LTR retrotransposons and retroviruses is the presence of theenvelope (env) gene in the latter, downstream of thepol gene. Theenv gene is involved in their infectious capacity. Here we report the presence ofenv-like sequences in the genome ofQuercus suber (cork oak), one of the most economically important Portuguese species. These gene sequences were isolated through DNA amplification betweenRNaseH conserved motifs and 3’ LTR, based on the structure ofcopia retrotransposons. Phylogenetic analysis revealed that almost all the clones isolated are clustered withCyclops-2, aTy3-gypsy element identified inPisum sativum, except one clustered withgypsy andcopia retroelements found in different species. This suggests the existence of a potential ancestral sequence of theenv gene, prior to the separation ofTy3-gypsy andTy1-copia retrotransposons. Additionally, the isolatedenv-like sequences showed 26–39% of homology withenv-like sequences characterized in viruses. The origin ofenv-like sequences in retrotransposons from host plant taxa is discussed.     

SIRE-1, A putative plant retrovirus is closely related to a legume TY1-copia retrotransposon family

SIRE-1 is a potential soybean retrovirus which has a gene order similar to Ty1-copia retrotransposons but also contains an envelope-like open reading frame (ORF), which is characteristic of retroviruses. PCR and Southern analysis reveals that SIRE-1 is closely related to a legume-wide family of envelope-lacking Ty1-copia group retrotransposons which suggests that SIRE-1 was formed by the recent acquisition of an envelope gene by a Ty1-copia retrotransposon.     

Genetic relationships between peanut and wild species ofArachis sect.Arachis (Fabaceae): Evidence from RAPDs

Twenty-six accessions of wildArachis species and domesticated peanuts,A. hypogaea, introduced from South America were analyzed for random amplified polymorphic DNA (RAPD). The objective of the study was to investigate inter- and intraspecific variation and affinities among species of sect.Arachis which have been proposed as possible progenitors for the domesticated peanut. Ten primers resolved 132 DNA bands which were useful for separating species and accessions. The most variation was observed among accessions ofA. cardenasii andA. glandulifera whereas the least amount of variation was observed inA. hypogaea andA. monticola. The two tetraploid species could not be separated by using RAPDs.Arachis duranensis was most closely related to the domesticated peanut and is believed to be the donor of the A genome. The data indicated thatA. batizocoi, a species previously hypothesized to contribute the B genome toA. hypogaea, was not involved in its evolution. The investigation showed that RAPDs can be used to analyze both inter- and intraspecific variation in peanut species. Southern hybridization of RAPD probes to blots containing RAPD of theArachis species provided information on genomic relationships and revealed the repetitive nature of the amplified DNA.