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1.
Ac/Ds标签系统与水稻功能基因组学 总被引:7,自引:0,他引:7
自2002年水稻基因组测序完成后,水稻功能基因组学研究正在成为水稻研究的重要内容.构建突变体库是研究功能基因组学的一条重要而有效的途径.利用外源的Ac/Ds (Activator/Dissociation)标签系统是构建插入突变体库较为理想的方法,经过多年发展完善,其在水稻中已有广泛的应用,但仍面临着一些需要解决的实际问题.文章对Ac/Ds标签系统的转座行为及其构建突变体库的问题和优点进行了综述,总结了近年来Ac/Ds标签系统在水稻中的研究进展,分析了利用Ac/Ds标签系统进行功能基因组学研究所面临的挑战. 相似文献
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Kuromori T Hirayama T Kiyosue Y Takabe H Mizukado S Sakurai T Akiyama K Kamiya A Ito T Shinozaki K 《The Plant journal : for cell and molecular biology》2004,37(6):897-905
More than 10 000 transposon-tagged lines were constructed by using the Activator (Ac)/Dissociation (Ds) system in order to collect insertional mutants as a useful resource for functional genomics of Arabidopsis. The flanking sequences of the Ds element in the 11 800 independent lines were determined by high-throughput analysis using a semi-automated method. The sequence data allowed us to map the unique insertion site on the Arabidopsis genome in each line. The Ds element of 7566 lines is inserted in or close to coding regions, potentially affecting the function of 5031 of 25 000 Arabidopsis genes. Half of the lines have Ds insertions on chromosome 1 (Chr. 1), in which donor lines have a donor site. In the other half, the Ds insertions are distributed throughout the other four chromosomes. The intrachromosomal distribution of Ds insertions varies with the donor lines. We found that there are hot spots for Ds transposition near the ends of every chromosome, and we found some statistical preference for Ds insertion targets at the nucleotide level. On the basis of systematic analysis of the Ds insertion sites in the 11 800 lines, we propose the use of Ds-tagged lines with a single insertion in annotated genes for systematic analysis of phenotypes (phenome analysis) in functional genomics. We have opened a searchable database of the insertion-site sequences and mutated genes (http://rarge.gsc.riken.go.jp/) and are depositing these lines in the RIKEN BioResource Center as available resources (http://www.brc.riken.go.jp/Eng/). 相似文献
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Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences 总被引:20,自引:0,他引:20
Kolesnik T Szeverenyi I Bachmann D Kumar CS Jiang S Ramamoorthy R Cai M Ma ZG Sundaresan V Ramachandran S 《The Plant journal : for cell and molecular biology》2004,37(2):301-314
A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds flanking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identified on chromosome 7 within a 40-kbp region. One-third of Ds flanking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), confirming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast artificial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the efficiency of this system is suitable for large-scale transposon mutagenesis in rice. 相似文献
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Distribution of T-DNA carrying a Ds element on rice chromosomes 总被引:3,自引:0,他引:3
WANG Jiang LI Lin WAN Xinshan AN Linsheng & ZHANG Jingliu National Key Laboratory of Plant Molecular Genetics Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China 《中国科学:生命科学英文版》2004,47(4):322-331
Rice is one of the most important crops in the world, and is widely studied as a model for cereal ge-nomics because of its small genome size (about 430 Mbp), and its colinearity at the sequence level with limited regions of other cereal genomes. In addition, there are a large number of rice databases document-ing molecular markers, genome sequences, EST se-quences and trait mutants[1—4]. Functional genomic studies of rice are increasing with the availability of the complete genome sequence. … 相似文献
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Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agrobacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent
transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA
and flanking sequence of rice genome. Type I sequences were the most common and showed canonical integration that T-DNA right
border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed
a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome
sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using
BLAST analysis. The 340 Ds insertions at an average interval of 0.8 megabase (Mb) constructed a basic framework of Ds starter
points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one
was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds
transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis. 相似文献
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Hang Gyeong Chin Sung Han Park Mi Sook Choe Su Hyun Park Byeong Keun Oh Gi Hwan Lee Hae Choon Choe Moo Je Cho Jong Chan Hong Chang-deok Han 《Journal of Plant Biology》2000,43(1):1-9
Wheat dwarf virus (WDV) is a monocot-infecting geminivirus that replicates in infected tissue as double-stranded DIMA. We
evaluated whether the WDV vector system bearingDs could be used as an effective insertional mutagen in rice. Molecular data showed thatDs was excised from WDV vectors once the WDV-carryingDs (WDV::Ds) and the genomicAc vector were co-introduced into rice calli. Mature TO and T1 transgenic plants were analyzed for the distribution and inheritance
ofDs inserts. Southern analysis indicated that theDs elements excised from WDV vectors were stably inserted into genomes. The number of transposedDs ranged from zero to three copies, among independent transformants. Meanwhile, untransposedDs (WDV::Ds) were present in multiple-copies in genomes. Southern analysis of the selfed progeny of T0 plants demonstrated that
most WDV::Ds were co-segregated among siblings. This indicated that these elements were integrated into the same single loci.
However, a fewDs were found to segregate independently from the majority ofDs. In this report, we discuss the efficiency of WDV vectors in generating multicopyDs in rice genomes. 相似文献
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Analysis of gene-trap Ds rice populations in Korea 总被引:1,自引:0,他引:1
Park SH Jun NS Kim CM Oh TY Huang J Xuan YH Park SJ Je BI Piao HL Park SH Cha YS Ahn BO Ji HS Lee MC Suh SC Nam MH Eun MY Yi G Yun DW Han CD 《Plant molecular biology》2007,65(4):373-384
Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes.
In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations
and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and
indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of
a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter
gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies
of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Sung Han Park, Nam Soo Jun, Chul Min Kim are contributed equally to this paper 相似文献
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不同启动子控制下Ac转座酶基因的表达对玉米Ds因子在水稻中切离频率的影响 总被引:2,自引:2,他引:2
用水稻愈伤组织比较了Ac启动子、35S启动子与Ubi启动子控制下Ac转座酶基因(Ts)的表达对Ds因子切离频率的影响。结果表明Ubi启动子与Ac转座酶编码区嵌合基因(Ubipro-Ts)反式激活Ds因子的切离频率最高,达到了72.9%。通过杂交将Ubipro-Ts基因导入Ds因子转化植株,得到9株Ubipro-Ts基因与Ds因子共存的F1代杂交水稻植株,其中有8株Ds因子发生了切离。用Inverse-PCR的方法从其中一株杂交植株中克隆到Ds因子的旁邻序列,其DNA顺序与亲本中Ds因子原插入位点的序列不同,表明Ds因子转座到了新的基因组位点。 相似文献
10.
利用Ac/Ds转座子系统在水稻中获得无选择标记转基因植株的方法 总被引:7,自引:0,他引:7
获得无选择标记转基因植株是进行重复转基因及消除转基因植株中标记基因潜在危害性的关键。实验采用了Ac/Ds转座子系统在水稻(Oryza sativa,L.)中进行无hpt选择标记的转基因。将含有目的基因bar的Ds元件和hpt标记基因置于同一个T-DNA中,通过农杆菌(Agrobacterium tumefaciens)EHAl05介导将Ac-T-DNA及Ds-T-DNA分别转入到不同的水稻植株,再将单拷贝的Ac-T-DNA植株与单拷贝的Ds-T-DNA植株杂交得到同时含有Ac和Ds元件的F1植株,Fl自交产生F2后代,F2植株中转座后的Ds元件与T-DNA独立分离,在总共100株F2水稻植株中筛选得到2株只含有Ds元件插入而无hpt标记基因的转基因水稻植株。结果表明,利用Ac/Ds转座子系统在水稻中获得无选择标记的转基因植株是可行的。 相似文献
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利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略. 相似文献
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Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析 总被引:9,自引:0,他引:9
利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7%。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。 相似文献
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Raina Surabhi Mahalingam Ramamurthy Chen Fuqiang Fedoroff Nina 《Plant molecular biology》2002,50(1):91-108
Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis. 相似文献
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Transposon tagging in rice 总被引:28,自引:0,他引:28
Izawa Takeshi Ohnishi Tohru Nakano Toshitsugu Ishida Nobuhiro Enoki Hiroyuki Hashimoto Hisako Itoh Kimiko Terada Rie Wu Chuanyn Miyazaki Chikara Endo Tomoko Iida Shigeru Shimamoto Ko 《Plant molecular biology》1997,35(1-2):219-229
To develop an efficient gene isolation method for rice we introduced the maize Ac/Ds system into rice. Extensive analysis of their behavior in rice for several generations indicated that Ac and Ds in the presence of Ac transposase gene actively transpose in rice. A wide spectrum of mutations affecting growth, morphogenesis, flowering time and disease resistance have been obtained in the population carrying Ac/Ds and some of them were genetically analyzed. Main efforts are currently being made to isolate genes responsible these mutations. In addition, a number of Ac/Ds were mapped on chromosomes and mapped elements will be used in the future for directed tagging of genes with known chromosomal positions. 相似文献
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Wu C Li X Yuan W Chen G Kilian A Li J Xu C Li X Zhou DX Wang S Zhang Q 《The Plant journal : for cell and molecular biology》2003,35(3):418-427
Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T1 families corresponded well with the T0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio. It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice. 相似文献
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Dong-Soo Park Soo-Kwon Park Sang-Ik Han Hoe-Jeong Wang Nam-Soo Jun Norvie L. Manigbas Young-Min Woo Byoung-Ohg Ahn Doh-Won Yun Ung-Han Yoon Yong-Hwan Kim Myung-Chul Lee Doh-Hoon Kim Min-Hee Nam Chang-Deok Han Hang-Won Kang Gihwan Yi 《Molecular breeding : new strategies in plant improvement》2009,24(1):1-15
A gene detection strategy using two-component Ac/Ds construct, with the mobile Ds transposon, has been developed to better understand gene functions in crops. Currently, 115,000 Ds insertion lines have been generated through the Ac/Ds gene trap system in Korea using japonica rice Dongjin as donor. Four hundred and thirty-seven mutants from 12,162 Ds-tagged lines were catalogued, including physiological and agronomic traits. Different traits were identified with distinct
characteristics in terms of tillers, panicles, leaves, flowers, seed, chlorophyll content, and height. Culm and panicle length,
number of panicles, and days to flowering of the Dongjin Ds population revealed high standard deviations compared with the donor cultivar. An evaluation of the Ds distribution on the chromosome revealed that 74.5% of the Ds were reinserted into gene-rich regions, making this Ac/Ds-mediated gene trap system useful in helping to gain an understanding of the function of genes and thus improve the gene-tagging
system in rice. 相似文献