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.     

BAC文库构建中的几个技术问题探讨

构建BAC文库有两个关键环节：一是载体的克隆;二是HMW（high molecular weight)DNA的制作。针对这两个问题,作者在载体的纯化及基因组DNA的回收选择等方面做了较深入的探讨。     

白氏文昌鱼单个体基因组BAC文库的构建

隶属于头索动物亚门的文昌鱼是现存生物中最近似于脊椎动物亚门直接祖先的一个类群, 具有重要的进化地位, 是研究脊椎动物原始祖先的重要材料和模式动物。随着文昌鱼实验室连续繁殖的成功, 全基因组测序成为中国文昌鱼模式化急需完成的工作之一。文章从单条雄性白氏文昌鱼精巢组织中提取高质量的基因组DNA, 经EcoRⅠ限制性内切酶和EcoRⅠ甲基化酶酶切, 脉冲场电泳选择合适酶切DNA片段, 连接线性磷酸化的载体pCC1BAC, 转化大肠杆菌EPI300 E. coli, 构建了含有44 706个克隆的全基因组BAC( Bacterial artificial chromosome)文库, 该文库平均插入片段80 kb,具有9倍的基因组覆盖率, 基本能够满足功能基因等研究需要, 为中国文昌鱼全基因组测序打下基础。     

新疆军垦型细毛羊基因组BAC文库的构建

选用新疆军垦型细毛羊为材料,.构建了含有190 464个克隆的BAC文库,文库平均插入片段大小为133 kb,同时文库92.5%的克隆插入片段大于100 kb,而且有部分克隆甚至大于300 kb,这将满足大多数基因筛选的要求.假定绵羊的基因组含有3x106 kb,根据文库的平均插入片段大小,计算的文库基因组覆盖率为8倍.因此,从文库筛选到目的片段的概率为98.2%.由于该文库中插入的外源片段来自新疆军垦型细毛羊的基因组,这对于研究新疆军垦型细毛羊的特殊性状的基因与其他绵羊品种和物种之间的差异,及构建其全基因组物理图谱和基因图谱地完善是非常有利的.     

PCR筛选BAC文库和直接BAC末端测序方法的建立

建立了一种用PCR方法筛选富含高度重复序列的大麦BAC DNA 文库和直接对 BAC DNA进行末端测序的方法.用PCR技术进行大麦BAC DNA 文库(含816个平板,每个平板含384个克隆)的筛选分4步进行.在实验中,建立了两个水平的BAC DNA池(一级池和二级池).一个二级池由一个平板(含有384个克隆)的DNA 组成,一个一级池由连续10个稀释100倍的二级池的DNA混合而成(如1～10,11～20等),共82个一级池.BAC DNA 文库筛选的第一步是对82个一级池的筛选.得到阳性一级池后(如2号一级池),对其所含的10个二级池(从11～20)进行第二步筛选.得到阳性二级池后,培养相应的阳性平板的所有克隆(384个),从头开始(左上侧),每相邻的4个克隆为一组,在96孔板上(4 X 96=384) 进行第三轮PCR反应;之后对筛选结果为阳性的4个克隆分别进行菌落 PCR(第四轮)得到单一阳性克隆.根据BAC DNA Hind III 酶切指纹图谱,对同一引物筛选的BACs进行重叠群作图(Contig).对代表contig 的两端的BAC DNA直接进行末端测序并对测序结果Blast,以检测其在大麦中是否属于单拷贝序列.根据测序和Blast结果设计引物,用中国春附加系(附加大麦染色体)对来自BAC克隆的引物进行染色体定位并用分离群体进行遗传学作图,以确定是否可以用作下一步的染色体步行.     

以色列野生二粒小麦(Triticum dicoccoides)耐盐性鉴定

采用水培法对从以色列引进的分属8个自然群体的93份野生二粒小麦进行了耐盐性初步鉴定.结果表明:①供试材料中仅有高耐盐材料3份,耐盐材料7份 ,耐盐性较好的材料仅占参试材料总数的10.7%;②8个自然群体的野生二粒小麦的耐盐性存在较大差异,其中3号群体耐盐性明显高于其他群体,说明来自Tabigha地区的野生二粒小麦具有丰富的耐盐基因源;③将经过鉴定的耐盐性较好的野生二粒小麦与普通小麦中的耐盐品种进行比较研究发现,以色列耐盐性较好的野生二粒小麦的耐盐性不如普通小麦中的茶淀红和SW10两个耐盐品种.     

山羊瘤胃微生物宏基因组BAC文库的构建与分析

从山羊瘤胃液中提取混合微生物DNA,经BamHI部分酶切得到50kb～800kb的DNA片段后,将其连接到pCCIBAC载体上,转化E．coliEPI300,建立山羊瘤胃微生物BAC文库。经RFLP鉴定分析,该文库12672个克隆,平均插入片段为6lkb。该文库的构建为后续新型基因的筛选提供了材料,为进一步研究山羊瘤胃微生物奠定了基础。     

一种存放大基因组文库的新方法

用野生一粒小麦为材料,以细菌人工染色体（pECBAC1)为载体构建了细菌人工染色体克隆混合池（Bacterial artificial chromosome pool）,每池100个克隆。经初步验证,池中靶克隆经12h的培养后仍稳定存活。克隆之间的竞争实验仍在进行当中。     

淀粉酶链霉菌BAC基因组文库的构建及鉴定

提取淀粉酶链霉菌SM33基因组DNA,用Hind III部分酶解后回收40 kb～60 kb大小的高分子量DNA,与质粒载体pIndigoBAC536连接,电击转化EPI300感受态细胞,经蓝白斑筛选共挑取了5 184个白色克隆.从文库中随机挑选10个克隆,酶切检测平均插入片段约为50 kb,覆盖了32.4倍基因组.并且插入片段均具有9～15个Not I酶切位点,符合链霉菌基因组特征.通过对BAC文库的筛选,获得苹果酸脱氢酶基因(mdh)的保守序列,与已知的链霉菌mdh具有很高的相似性.淀粉酶链霉菌BAC基因组文库的建立,对基因克隆、基因组物理图谱、次级代谢途径、新抗生素的发现以及工业用酶的应用等研究均有重要意义.     

细菌人工染色体文库的构建及应用

细菌人工染色体（BAC）是第二代大片段DNA的克隆载体系统,具有容量大、嵌合率低、遗传特性稳定、转化效率高、插入片段易回收、操作简便等优点,因而被广泛应用于基因组较大的真核生物基因组研究中,并发挥着前所未有的重要作用。本文综述了BAC的发展,利用此载体构建基因组文库的程序和鉴定方法,及其在物理图谱构建、图位克隆、基因组测序、转基因技术等研究中的应用。     

细菌人工染色体基因组文库构建方法的改进

目的:建立一种改进的更简便、易操作的细菌人工染色体(BAC)文库构建方法。方法:在构建猪霍乱沙门氏菌基因组大片段DNA的BAC文库时,对改进的基因组BAC文库构建方法和常规的BAC文库构建方法进行比较。结果:利用改进的方法可简便快速地构建猪霍乱沙门氏菌基因组BAC文库。结论:使用2种方法构建BAC文库,其转化效率,以及在BAC克隆中插入的DNA片段的大小和BAC克隆的稳定性等都相同,从而表明改进的方法更简单、更方便,它能使BAC文库的构建更为高效。     

Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

Although the centromeres of some plants have been investlgated prevlously, our knowledge of the wheat centromere Is still very llmlted. To understand the structure and functlon of the wheat centromere, we used two centromeric repeats （RCS1 and CCS1-5ab） to obtain some centromere-assoclated bacterial artificial chromosome （BAC） clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat （Triticum boeoticum Boiss.; 2n=2x=14） BAC library. Southern hybridization results indicated that, of the 32 candidates, there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization （FISH） analysis Indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromerlc regions in hexaplold wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or Dgenome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific （or rich） sequences dispersing on chromosome arms in the BAC clone TbBACS. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation In the period before metaphase.     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Construction of a Bacterial Artificial Chromosome Library and Characterization of hrp/hrc Gene Cluster of Pseudomonas Syringae Pathovar tagetis LMG5090

Pseudomonas syringae pv. tagetis causes apical chlorosis of several plant species in the Asteraceae, including marigold. As a means to facilitate the isolation of pathogenicity genes and to characterize the genome of this bacterium, we have constructed a bacterial artificial chromosome library of P. syringae pv. tagetis strain LMG5090. The library consists of 1,536 clones with insert size ranging from 30 to 160 kb and an average size of 86 kb. Based upon colony hybridization, the BAC clone 420E23 containing the hrp/hrc gene cluster encoding the type III secretion system was identified from this library and subsequently shotgun sequenced. The hrp/hrc gene cluster of P. syringae pv. tagetis has a 23 kb sequence which contains 27 open reading frames. Comparative analysis of the hrp/hrc gene cluster of P. syringae pv. tagetis LMG5090, P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, and P. syringae pv. phaseolicola 1448A revealed that the entire hrp/hrc gene cluster of P. syringae pv. tagetis is conserved and identically arranged in all four pathovars     

Genomic Bacterial Artificial Chromosome Library of the Japanese Flounder Paralichthys olivaceus

We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC-lac vector. This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells. The average size of inserted DNA was calculated to be 165 kb. The BAC library was determined to cover 9 times the Japanese flounder haploid genome. The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis. Received March 1, 2000; accepted May 29, 2000.