小麦-簇毛麦6VS/6AL易位系可转化人工染色体(TAC)文库的构建

可转化人工染色体(Transformation\|competent Artificial Chromosome,TAC)是具有克隆和转移大片段基因能力的新型载体,是植物基因克隆和转化的有效工具。为了克隆小麦抗白粉病基因和其它基因,本研究用TAC载体pYLTAC17构建了带有抗白粉病基因Pm21的小麦簇毛麦6VS/6AL易位系的基因组DNA文库。该文库包含210万个克隆,平均插入片段35kb,库容相当于普通小麦基因组的49倍。本文库以约1000个克隆组成1个混合池的方式保存在22块96孔平板,可用PCR方法进行文库的筛选。     

东乡野生稻双元细菌人工染色体(BIBAC)文库的构建

双元细菌人工染色体(binarybacterialartificialchromosome,BIBAC)是能直接将大片段DNA转入植物的载体,是植物基因图位克隆和构建植物基因嵌入突变体库的重要工具。该研究以东乡野生稻为材料,构建其BIBAC文库。该文库由14592个克隆组成,平均插入片段大小为65kb,覆盖率为2倍基因组。稳定性检测结果表明,东乡野生稻基因组DNA能够在BIBAC载体中稳定存在。     

抗黄矮病小麦-中间偃麦草易位系基因组可转化人工染色体文库的构建及初步筛选

利用抗黄矮病小麦 -中间偃麦草易位系HW6 4 2的细胞核DNA构建了一个可转化人工染色体 (transformation competentartificialchromosome,TAC)文库 ,文库由 2 .3× 10 6 克隆构成 ,重组率为 90 .4 8% ,平均插入片段大小为 2 2kb左右 ,约覆盖普通小麦单倍体基因组 2 .5倍 ,在该文库中分离得到单拷贝DNA序列的几率约是 95 .77%。文库保存在 2 4块 96孔板中 ,每个孔中约含有 10 0 0个不同的重组克隆 ,可以采用PooledPCR的方法对文库进行筛选。用来源于小麦的简单重复序列 (simplesequencerepeat,SSR)引物wms37扩增中间偃麦草、抗病易位系及感病材料 ,得到一条与抗性共分离的特异条带 ,约 4 5 0bp。将此特异标记条带转化为SCAR(sequencecharacterizedamplifiedregion)标记 ,用于筛选HW6 4 2基因组TAC文库 ,得到 12个阳性克隆。对阳性克隆进行了PCR Southern验证 ,以中间偃麦草基因组总DNA为探针与限制酶HindⅢ消化后的阳性克隆杂交 ,其中 10个阳性克隆分别有 1～ 6条杂交带 ,结果表明 ,这 10个阳性克隆可作为抗黄矮病相关基因筛选的候选克隆     

可转化人工染色体文库的构建与鉴定

随着人类和植物基因组计划的实施,能容纳大片段的人工染色体载体发展迅速。而用YAC、BAC和PAC等基因组文库进行目的基因的筛选,在获得候选克隆后,通常要进行亚克隆,然后对每个亚克隆逐一进行基因功能互补试验,不仅工作量大,而且有遗漏目的基因的危险。TAC载体含P1质粒和Ri质粒的复制子,可直接转化植物,大大加速了工作进程。概括性的叙述了TAC载体的发展,TAC文库构建的程序及文库鉴定的问题。     

基因组细菌人工染色体文库(BAC)的构建及应用

细菌人工染色体 (BAC)是一种承载DNA大片段的克隆载体系统 ,用于人、动物和植物基因组文库构建。BAC具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点。BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础 ,可用于真核生物重要基因及全基因组物理作图、重要性状基因的图位克隆、基因结构及功能分析。本文主要综述了细菌人工染色体的构建与其鉴定 ,及其在物理图谱构建、图位克隆、转基因技术等研究上的应用。     

野生一粒小麦BAC文库的构建和鉴定

以细菌人工染色体pECBAC1为载体,构建了野生一粒小麦(Triticum boeoticum B oiss)的基因组BAC文库.该文库共包含约17万个克隆,平均插入片段长度为104 kb,按野生一粒小麦基因组为5 600 Mb计算,文库覆盖了约3倍的该物种基因组.用大麦叶绿体psb A基因和玉米线粒体atp6基因作混合探针,检测发现该文库中含细胞器基因组同源序列的克隆数小于1% .该文库的建成,为小麦基因的克隆及基因组学研究提供了技术平台.     

细菌人工染色体的研究和应用

细菌人工染色体 (Bacterialartificialchromosome ,BAC)是第二代大片段DNA的克隆载体系统。因其嵌合率低 ,遗传稳定性好 ,重组DNA容易分离和制备 ,转化效率高等 ,弥补了YAC的不足 ,很快在基因组研究中处于中心地位。近年来 ,已有多种BAC载体被构建出来 ,这些BAC载体在复杂基因组大片段文库的构建 ,基因的图位克隆 ,基因组物理图谱的构建 ,基因和基因组测序 ,基因组织结构分析 ,染色体组织和进化 ,以及基因的遗传转化和调控研究中得到了广泛的应用。     

藏鸡微卫星文库的构建与微卫星标记筛选

通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析.将藏鸡基因组DNA经Sau3AI酶切后纯化回收,连接特定接头.用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200～900 bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库.从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性.实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率.     

野生一粒小麦BAC文库的构建和鉴定

以细菌人工染色体pECBAC1为载体 ,构建了野生一粒小麦 (TriticumboeoticumBoiss)的基因组BAC文库。该文库共包含约 17万个克隆 ,平均插入片段长度为 10 4kb ,按野生一粒小麦基因组为 5 6 0 0Mb计算 ,文库覆盖了约 3倍的该物种基因组。用大麦叶绿体psbA基因和玉米线粒体atp6基因作混合探针 ,检测发现该文库中含细胞器基因组同源序列的克隆数小于 1%。该文库的建成 ,为小麦基因的克隆及基因组学研究提供了技术平台     

雨生红球藻基因组文库的构建及鉴定

本研究以雨生红球藻34-1n为材料,提取其基因组DNA,利用限制性内切酶Sau3AⅠ对基因组DNA进行酶解,回收6~8kb的基因组DNA片段,并浓缩至200ng/μL。该片段与经BamH Ⅰ酶切和去磷酸化处理后的pUC18载体连接,然后电击转化到受体菌Escherichia.coli DH5α中,获得雨生红球藻34-1n的基因组文库。该文库的平均插入片段长度约为6.5kb,获得6×105个克隆数。通过PCR筛选,由雨生红球藻基因组文库中获得含bkt1序列的单克隆菌,与β-胡萝卜素氧化酶序列(GenBank:DQ086233.1)进行比对,结果表明bkt1基因组序列含有6个外显子。本研究为进一步鉴定雨生红球藻相关基因提供了一个文库平台。     

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.     

可转化人工染色体(TAC)文库载体DNA的制备

对可转化人工染色体(TAC)pYLTAC747NH/sacB文库载体DNA的制备条件进行了较系统的模索和研究.结果表明,该文库载体经碱裂解法提取、QIAGEN Plasmid Mini kit纯化后,可获得较纯净的载体DNA.对其闭环载体DNA分别用不同酶量的Hinid Ⅲ酶切处理,经琼脂糖凝胶电泳检测得出其最佳Hind Ⅲ完全酶切条件为2 U Hind Ⅲ/μg闭环载体DNA、37℃酶切30 min;分别用0.5 MBU和1 MBU HK脱磷酶/μg对其线性载体DNA进行脱磷处理,经电泳和载体自连产物电转化检测表明其适宜的完全脱磷条件为1 MBU HK脱磷酶/μg线性载体DNA,30℃脱磷1 h;将所制备的线性载体DNA与λ DNA/Hind Ⅲ酶切片段进行连接,连接产物转化频率较高,其电转化大肠杆菌DH10B感受态细胞频率可达到9.6×10s.     

Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat.

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276,480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720,384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.     

Development of a new transformation-competent artificial chromosome (TAC) vector and construction of tomato and rice TAC libraries

Recent research has shown that BIBAC (binary bacterial artificial chromosome) and TAC (transformation-competent artificial chromosome) vector systems are very useful tools for map-based cloning of agronomically important genes in plant species. We have developed a new TAC vector that is suitable for both dicot and monocot transformation. Using this new TAC vector, we constructed large-insert genomic libraries of tomato and rice. The tomato library contains 96,996 clones (28.3-38.5 kb insert size) and has 3.18 haploid genome equivalents. The rice TAC library has 32.7 kb average insert size and has 9.24 haploid genome equivalents. The quality of these two libraries was tested using PCR to verify genome coverage. Individual clones were characterized to confirm insert integrity by Southern analysis, end sequencing and genetic mapping. To investigate the potential application of these TAC libraries in map-based cloning, TAC constructs containing a 45 kb fragment were introduced into the rice genome via Agrobacterium-mediated transformation. Molecular analysis indicates that the 45 kb fragment was successfully transferred into the rice genome. Although rearrangements of the introduced DNA were detected, 50% of regenerated plants contained at least one intact copy of the 45 kb clone and associated vector sequences. These libraries provide us with a valuable resource to rapidly isolate important genes in tomato and rice.     

Structural analysis of a Lotus japonicus genome. V. Sequence features and mapping of sixty-four TAC clones which cover the 6.4 mb regions of the genome.

We determined the nucleotide sequences of 64 TAC (transformation-competent artificial chromosome) clones selected from genomic libraries of Lotus japonicus accession Miyakojima MG-20 based on the sequence information of expressed sequence tags (ESTs), cDNAs, genes and DNA markers from L. japonicus and other legumes. The length of the DNA regions sequenced in this study was 6,370,255 bp, and the total length of the L. japonicus genome sequenced so far is 32,537,698 bp together with the nucleotide sequences of 256 TAC clones previously reported. Five hundred forty-eight potential protein-encoding genes with known or predicted functions, 127 gene segments and 224 pseudogenes were assigned to the newly sequenced regions by computer prediction and similarity searches against the sequences in protein and EST databases. Based on the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was genetically localized onto the linkage map of two accessions of L. japonicus, MG-20 and Gifu B-129. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Chromosomal map of the model legume Lotus japonicus

Lotus japonicus is a model plant for the legume family. To facilitate map-based cloning approaches and genome analysis, we performed an extensive characterization of the chromosome complement of the species. A detailed karyotype of L. japonicus Gifu was built and plasmid and BAC clones, corresponding to genetically mapped markers (see the accompanying article by Sandal et al. 2002, this issue), were used for FISH to correlate genetic and chromosomal maps. Hybridization of DNA clones from 32 different genomic regions enabled the assignment of linkage groups to chromosomes, the comparison between genetic and physical distances throughout the genome, and the partial characterization of different repetitive sequences, including telomeric and centromeric repeats. Additional analysis of L. filicaulis and its F(1) hybrid with L. japonicus demonstrated the occurrence of inversions between these closely related species, suggesting that these chromosome rearrangements are early events in speciation of this group.     

用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆

用根据抗病基因保守区设计的一对简并性引物,从小麦－簇毛麦易位系6VS／6AL cDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点（Nucleotide binding site,NBS）结构特点的DNA片段克隆N7。从小麦－簇毛麦易位系6VS／6AL基因组TAC（Transformation-competent artificial chromosome,TAC）文库的22块96孔板提取所有2112个克隆池（每个池含约1000个克隆）的质粒,再根据N7的核苷酸序列设计一对特异引物,用克隆池PCR（pooled PCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。研究结果表明克隆池PCR法对克隆数目巨大的基因组文库的筛选很有效。     

稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

Construction,characterization, and screening of a transformation-competent artificial chromosome library of peach

As a genome model of fruit trees, peach (Prunus persica [L.] Batch) has advantages for studying structural and functional genomics. Okubo, a traditional peach variety used as a parent in Asian peach breeding, displays economically valuable agronomic traits. To develop an efficient platform for peach gene cloning and genomic research, a large-insert genomic DNA library of Okubo was constructed in a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, which can accept and stably maintain large genomic DNA fragments in bothEscherichia coli andAgrobacterium tumefaciens. The TAC library contains 41,472 recombinant clones with an average insert size of approximately 42 kb, and it is equivalent to 6 haploid peach genomes. The TAC library was stored in 2 ways: one copy as frozen cultures in 108 pieces of 384-well plates and another copy as bulked pools in 36 pieces of 96-well plates, each well containing 12 individual clones. The lack of hybridization signal to chloroplast and mitochondrial genes indicated that the TAC library had no significant cytoplast organelle DNA contamination. TAC clones were stable inE. coli cells until generation 100 and stable in bothE. coli andA. tumefaciens. Twenty-one clones containing the polygalacturonase-inhibiting protein (PGIP) gene were detected by using pooled PCR in the TAC library. Positive clones can be used for peach PGIP gene cloning and functional analysis. The library is well suited for gene cloning and genetic engineering in peach.     

Structural analysis of a Lotus japonicus genome. IV. Sequence features and mapping of seventy-three TAC clones which cover the 7.5 mb regions of the genome.

Using the sequence information of expressed sequences tags (ESTs), cDNAs and genes from Lotus japonicus and other legumes, 73 TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of L. japonicus accession MG-20, and their nucleotide sequences were determined. The length of the DNA sequenced in this study was 7,455,959 bp, and the total length of the DNA regions sequenced so far is 26,167,443 bp together with the nucleotide sequences of 183 TAC clones previously reported. By similarity searches against the sequences in protein and EST databases and prediction by computer programs, a total of 699 potential protein-encoding genes with known or predicted functions, 163 gene segments and 267 pseudogenes were assigned to the newly sequenced regions. Based oil the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was located onto the linkage map of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.