番茄RAPD分析有效引物3′端序列的偏倚分布及其与基因组序列结构的关系

对番茄RAPD分析的有效引物与Operon引物系列（OP系列）的3′端序列作了统计比较,研究结果指出,翻茄有效引物系列不是OP系列的随机样本,而是3端序列的种类和分布上有偏倚性的选择性样本,虽然两个系列的引物都具有3’端同序现象,但对其分布频率正交比较结果差异极显,此类同序性3’端在番茄基因组的RAPD座位中是普遍分布 的,RAPD引物3’端序列的非随机分布,反映了番茄基因组内的多态性区段侧翼具有序列的特异性,这种特异性结构应是番茄基因组序列的一种属性,这种属性为番茄属基因组所共有。     

小麦及其近缘种中基因组特异性DNA重复序列的研究进展

本文对小麦族植物中基因组特异性DNA重复序列的分类、基本特征、分离和鉴定方法、在小麦遗传改良中的应用以及未来研究的发展趋势进行了简述。综合已有的研究结果可以看出基因组特异性DNA重复序列是小麦族植物基因组特异性形成的重要构成部分。对基因组特异性DNA重复序列的研究是认识小麦族植物基因组的有效途径之一,基因组特异性DNA重复序列的应用将进一步促进小麦族植物分子细胞遗传学和普通小麦遗传改良研究的进展。 Advances in Studies of Genome-Specific Repetitive DNA Sequences in Wheat and Related Species BAI Jian-rong1,2,JIA Xu1,WANG Dao-wen1 1.The State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,The Chinese Academy of Sciences,Beijing 100101,China; 2.Crop Genetics Institute,Shanxi Academy of Agricultural Sciences,Taiyuan 030031,China Abstract:In this paper we review recent advances in studies of several aspects of genome specific repetitive DNA sequences in wheat and related species.The available results demonstrate that genome specific repetitive DNA sequences are important components of genome specificity in wheat and related species.Research on genome specific repetitive DNA sequences is essential to the elucidation of genome function.The application of genome specific repetitive DNA sequences will aid molecular cytogenetic studies in wheat and related species and contributes to genetic improvement of common wheat. Key words：wheat;genome specific repetitive DNA sequence;chromosome     

HIV-1融膜肽的编码DNA序列比较

通过分析GenBank中的全部95个HIV-1完整基因组序列,设计融膜肽"探针序列",对所获得的95段融膜肽的编码DNA序列进行了翻译、对准和分析.得到融膜肽及其编码序列的"优势序列"及突变分布。 Abstract:Fusion peptide coding DNA sequences were retrieved from 95 HIV-1 complete genome entries of GenBank.Results of translation,alignment and analysis led to "the dominant sequence" and the mutation distribution of the fusion eptide coding DNA sequences.     

ISSR分子标记及其在植物遗传学研究中的应用

ISSR分子标记是在SSR标记基础上发展起来的一种新技术,其基本原理是在SSR的5′或3′端加锚1~4个嘌呤或嘧啶碱基,然后以此为引物,对两侧具有反向排列SSR的一段基因组DNA序列进行扩增。重复序列和锚定碱基是随机选择的,扩增产物经聚丙烯酰胺或琼脂糖凝胶电泳分离后,每个引物可以产生比RAPD方法更多的扩增片段,因此,ISSR标记是一种快速、可靠、可以提供有关基因组丰富信息的DNA指纹技术。ISSR标记呈孟德尔式遗传,在多数物种中是显性的,目前已广泛用于植物品种鉴定、遗传作图、基因定位、遗传多样性、进化及分子生态学研究中。 ISSR Markers and Their Applications in Plant Genetics WANG Jian-bo Key Laboratory of MOE for Plant Developmental Biology,Wuhan University,Wuhan 430072,China Abstract:Recently,inter-simple sequence repeat (ISSR) markers have emerged as an alternative system with reliability and advantages of microsatellites (SSR).The technique involves amplification of genomic segments flanked by inversely oriented and closely spaced microsatellite sequences by a single primer or a pair of primers based on SSRs anchored 5′ or 3′ with 1-4 purine or pyramidine residues.The sequences of repeats and anchor nucleates are arbitrarily selected.Coupled with the separation of amplification products on a polyacrylamide or agarose gels,ISSR amplification can reveal a much larger number of fragments per primer than RAPD.It is concluded that ISSR technique provides a quick,reliable and highly informative system for DNA fingerprinting.ISSR markers are inherited in Mendelin mode and segregated as dominant markers.This technique has been widely used in the studies of cultivar identification,genetic mapping,gene tagging,genetic diversity,evolution and molecular ecology. Key words：molecular markers; ISSR; plant;applications     

利用RACE技术扩增大豆抗病基因同源cDNA 5′末端序列

利用抗病基因保守序列筛选大豆cDNA文库,获得一抗病基因同源cDNA片段,命名为KR3-1。根据KR3-1设计两个基因特异引物（GSP 和 NGSP）,分别与通用引物(UPM)和巢式通用引物（NUP）共同扩增,成功地克隆到了该基因的5′末端序列。该扩增片段长447 bp,与已知序列重叠部分为129 bp。 Abstract:Based on part of a known partial cDNA sequence of a disease resistance gene homolog,KR3-1,obtained by screening a cDNA library from soybean,5′-RACE-PCR was carried out with gene specific primers and universal primers.After the nested PCR reaction,an amplified fragment of 447 bp in length which overlapped the known KR3-1 sequence by 129 bp was obtained subsequently.Thus,a 5′ cDNA end of KR3 was successfully cloned.     

微卫星序列及其应用

微卫星序列广泛存在于各类真核生物基因组中,一般为散在分布的中等程度重复序列。不同物种中,微卫星序列的含量以及占优势的微卫星序列类型各不相同。复制时,微卫星序列易于发生长度突变,这种突变与微卫星序列的复制滑移有关,同时也受多种因素的影响。微卫星序列可能是原微卫星序列通过复制滑移使序列长度扩增形成的。进化过程中,微卫星序列的长度变化维持在一定的范围内。由于微卫星标记多态性高、重复性好,并且操作简单,因此在基因的定位、人类疾病诊断及预测、亲权分析、品种鉴定、进化研究,以及动植物分子标记辅助选择育种研究等领域中都有着重要的应用价值。 Abstract:Microsatellites,simple sequence repeats (SSR),are abundant and distributed throughout the eukaryote genome.The contents of microsatellites are variant in different creatures.There are also different types of microsatellites,which are dominant in different creatures.One of the most noticeable characters of microsatellites is that they are easy to expand during DNA replication.It is thought to attribute to DNA slippage.This kind of mutation is affected by many factors.It is guessed that microsatellites come from pro-microsatellites,while the pro-microsatellites origin from random point mutations.The length of microsatellites can be maintained under relative conservative ranges during species evolution.As they are abundant,codominatnt,distributed over the euchromatic part of the genome,and have the character of highly polimorphic,microsatellites are useful tools for gene mapping,clinical diagnosis and predicting,paternity or pedigree analysis,evolution study,and marker-assisted breeding.     

Analysis of collinear regions of Oryza AA and CC genomes

Comparative analyses of genome structure and sequence of closely related species have yielded insights into the evolution and function of plant genomes. A total of 103,844 BAC end sequences delegated -73.8 Mb of O. officinalis that belongs to the CC genome type of the rice genus Oryza were obtained and compared with the genome sequences office cultivar, O. sativa ssp.japonica cv. Nipponbare. We found that more than 45% of O. officinalis genome consists of repeat sequences, which is higher than that of Nipponbare cultivar. To further investigate the evolutionary divergence of AA and CC genomes, two BAC-contigs of O. officinalis were compared with the collinear genomic regions of Nipponbare. Of 57 genes predicted in the AA genome orthologous regions, 39 had orthologs in the regions of the CC genome. Alignment of the orthologous regions indicated that the CC genome has undergone expansion in both genic and intergenic regions through primarily retroelement insertion. Particularly, the density of RNA transposable elements was 17.95% and 1.78% in O. officinalis and O. sativa, respectively. This explains why the orthologous region is about 100 kb longer in the CC genome in comparison to the AA genome.     

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses （enJSRV）

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia （enJSRV-NM）, we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame （orf-x） that overlaps the 3＇ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain （Accession No.AF153615） and USA JSRV21 strain （Accession No. AF105220）. The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

一个水稻重复序列的分析与定位

在利用PCR简并引物扩增水稻NBS-LRR类抗病基因同源序列的研究中,克隆了一个大小为560 bp左右的重复序列,命名为DH17。序列分析和同源性比较发现,该序列包含352 bp的重复单位,与已报道的OS48和TrsA等重复单位序列进行比较,差异多低于5%, 具有很高的同源性,因此为同一重复序列家族。分子杂交表明,该序列在籼型品种"窄叶青8号"（ZYQ8）中以大量的串联拷贝存在,拷贝数显著高于粳型品种"京系17"（JX17）。利用ZYQ8和JX17组配的DH群体,通过 RFLP分析,直接将DH17的大量串联拷贝定位于ZYQ8的12号染色体长臂末端区域。 Abstract:A repeated sequence with a length of 560 bp,termed as DH17,was obtained during PCR amplification of rice NBS-LRR homologues.A repeated unit of 352 bp in the DH17 fragment was revealed through sequence analysis and comparison,which has a high homology with the known sequences of OS48 and TrsA,and belongs to the same repeat family.Southern hybridization displayed that there are higher DH17 copies in the genome of an indica variety,ZYQ8,than that in the genome of japonica variety,JX17.The tandom repeated DH17 sequence was mapped on the long arm end of chromosome 12 through RFLP analysis of a double haploid population derived from ZYQ8 and JX17 using DH17 as a probe.     

Complete nucleotide sequence and genome organization of tobacco mosaic virus isolated from Viciafaba

Based on reported TMV-U1 sequence, primers were designed and fragments covering the entire genome of TMV broad bean strain (TMV-B) were obtained with RT-PCR. These fragments were cloned and sequenced and the 5' and 3' end sequences of genome were confirmed with RACE. The complete sequence of TMV-B comprises 6 395 nucleotides (nt) and four open reading frames, which correspond to 126 ku (1 116 amino acids), 183 ku (1 616 amino acids), 30 ku (268 amino acids) and 17.5 ku proteins (159 amino acids). The complete nucleotide sequence of TMV-B is 99.4% identical to that of TMV-U1. The two virus isolates share the same sequence of 5', 3' non-coding region and 17.5 K ORF, and 6, 1 and 3 amino acid changes are found in 126 K protein, 54 K protein and 30 K protein, respectively. The possible mechanism on the infection of TMV-B in Vicia faba is discussed.     

SSR分子标记开发策略及评价

SSR标记以其多态性丰富、提供遗传信息多、操作便利并在基因组中分散分布等优点已成为最受人们欢迎的分子标记之一,在许多领域广泛应用。但SSR标记的主要缺点是首先要从该物种中获取重复序列两侧的序列信息,并设计引物,而后才能被利用。综述了几种有代表性的SSR标记开发策略,旨在为各物种SSR标记的开发提供参考信息。Abstract: SSR molecular markers have been used widely in many genetic studies and become one of the most popular molecular markers due to their high polymorphism, abundant informativeness, convenience of assay by PCR and distribution throughout the genome. The major drawback of SSR molecular markers is that their primers need to be designed according to the isolated sequence flanking the SSR from species that are being examined for the first time. The aim of the present paper is to review the several representative methods of SSR isolation described in the literature and provide useful information in making appropriate choices among the large number of currently available options.     

Repetitive Sequences in Plant Nuclear DNA:Types,Distribution, Evolution and Function

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150–400 base pairs(bp) in length.Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as ‘‘tuning knobs' ' in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences.     

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.     

T-DNA Integration Category and Mechanism in Rice Genome

T-DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T-DNA integration mechanism. The structures of T-DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T-DNA right border occurred mainly on five nucleotides “TGACA” in inverse repeat (IR) sequence of 25 bp, especially on the third base “A”. However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T-DNA right border, which made the T-DNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T-DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T-DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T-DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T-DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position of T-DNA. Some small regions in the fight border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the T-DNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T-DNA integration. The fragments flanking the T-DNA fight border in transformed rice plants could derive from different parts of the inner T-DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T-DNA, in which the homologous area was comparatively easier to be disrupted. The structure of flanking sequences of T-DNA integrated in the rice chromosome presented various complexities. These complexities were probably a result of different patterns of recombination in the integrating process. Some types of possible integrating mechanism are detailed.     

一步PCR快速扩增辽宁碱蓬甜菜碱醛脱氢酶cDNA 3'末端序列

根据已获得的辽宁碱蓬甜菜碱醛脱氢酶cDNA的部分序列,设计一条基因特异性引物,与通用引物并用,一步PCR成功地克隆了辽宁碱蓬甜菜碱醛脱氢酶cDNA 3′末端。与常规的3′RACE法相比,一步PCR法具有快速、简便、经济等优点,是一种非常快捷的扩增cDNA 3′末端序列的方法。 Abstract:Based on part of a known cDNA sequence of Suaeda liaotungensis betaine aldehyde dehydrogenase,we successfully cloned the 3′cDNA end of S.lianotungensis betaine aldehyde dehydrogenase using one step PCR with a gene specific primer and universal primer.Compared with the typical 3′ RACE,one step PCR is rapid,simple and inexpensive.It is very rapid to amplify an unknown cDNA 3′end using this method.     

Potyvirus属成员基因组全序列的简并引物PCR和RACE扩增方法

Based on multi-alignment of complete polyprotein amino acid sequences of genus Potyvirus,five degenerated primers were designedThey were Sprimer(5′-GGX AAY AAY AGY GGX CAZ CC-3′),pNIa(+)(5′-TNY TGG AAM CAY TGG AT-3′),pCI2(+)(5′-GCX ACX AAX ATX ATX GAX AA-3′),pCI1(+)(5′-GTX GGX TCX GGX AAX TCX AC-3′)and pHC(+)(5′-TGY GAY AAY CAZ TTX GA-3′)(X=A,T,C or G:Y=T or C;Z=A or G;N=A or T;M=A,T or G)Using degenerated PCR and modified RACE methods,a protocol for determination of complete genome sequence of potyviruses was established and proved to be successful on five potyviruses     

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Dear Editor, Recently, zinc finger nuclease, transcrip- tion activator-like effector nuclease, and RNA-guided Cas9 endonuciease （Cas9） have emerged as powerful means for genome editing （Conklin, 2013; Gaj eta[., 2013）. These nucleases are efficient in gen- erating double-strand breaks in the genome that can be repaired by error-prone nonho- mologous end joining leading to a functional knockout （KO） of the targeted gene or used to integrate a DNA sequence at a specific locus through homologous recombination.     

The complete genomic sequence of egg drop syndrome virus strain AAV-2

In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restrietion endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the eomplete genome nueleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on ganomic structure and the distribution of open reading frame (ORF). There are no elear E1, E3 and F4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding E1A, pⅤ and pⅨ, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group Ⅲ Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus