盐藻(Dunaliella viridis)细菌人工染色体文库的构建和鉴定

盐藻具有极强的耐盐能力,是研究植物耐盐机制的模式系统.为了对其耐盐机制进行深入的 研究,以pCC1BAC为载体,构建了盐藻的细菌人工染色体（BAC）文库.该文库共有9 216 个转化子,插入片段平均长度为55 kb,以单克隆形式保藏在96块96孔板中,并建立了四维P CR基因筛选体系,可以通过4轮PCR快速筛选获得阳性单克隆.根据本实验室分离到的两个盐 藻基因的cDNA序列（ DvSPT2和DvTPSP ）设计引物,通过PCR从该文库中各筛到4个阳性BA C克 隆,说明该文库能有效用于分离盐藻基因的基因组序列,据此推测该文库约覆盖4倍盐藻基 因组序列.     

稻瘟病抗性基因Pita2的克隆及其介导的防御相关基因表达分析

为了克隆位于水稻12号染色体着丝粒区域具有广谱抗性的稻瘟病抗性基因Pita2,研究构建并筛选了该基因供体品种PiNo.4的Fosmid文库,构建Pita2候选区域的物理图谱并互补验证了5个候选基因,克隆得到了稻瘟病抗性基因Pita2,确认了该基因就是Ptr基因.一组基因型接近、对Pita2致病性相反的稻瘟病菌菌株对水稻...     

水稻幼叶及幼穗cDNA文库的构建及初步分析

为了克隆水稻第6染色体上S5位点的基因及包括广亲和基因在内的重要功能基因,以水稻Oryza sativa L.广亲和品种Cpslo17为材料,以λTriplex2TM为载体利用SMART技术构建了幼叶和幼穗2种器官的cDNA噬菌体文库.幼叶和幼穗原始文库的克隆数目为别为1.5×105 pfu和2.45×105 pfu,插入片段的大小均在500～3 000 bp之间,文库的阳性克隆子的为别比例为97.0%和98.7%.此文库的建立为克隆广亲和基因和其它重要全长功能基因奠定了基础.     

黄瓜(Cucumis sativus L.) BAC文库的构建及连锁群特异克隆的分离

利用中国华北类型的黄瓜自交系S94构建一个BAC文库,该文库包含约19200个克隆,平均插入片段为105kb,大约覆盖黄瓜基因组的5倍．为了使黄瓜的遗传连锁群锚定其染色体,从黄瓜的7个连锁群共选择了22个标记,其中15个SCAR标记和7个SSR标记,利用这些标记用PCR的方法筛选BAC文库的DNA池,15个标记筛选到至少2个克隆,共筛选到60个BAC克隆,最后确定了22个BAC克隆作为连锁群特异克隆,这个BAC文库的构建为今后黄瓜基因组研究奠定了基础。     

Cupriavidus metallidurans CH34中2个苯酚降解基因簇的筛选与功能鉴定

Cupriavidus metallidurans(C.metallidurans)CH34是一种重金属耐受性细菌,能在以苯酚、甲苯酚、苯甲酸、苯胺等芳香族化合物为唯一碳源和能源的培养基中生长,其基因组中含有2个苯酚降解基因簇.以载体pIndigo-BAC 5构建C.metallidurans CH34的细菌人工染色体(bacterial artificial chromosome,BAC)文库,获得约3万个克隆,平均插入片段大小为30 kb,插入频率为98%,推测该文库覆盖CH34基因组约1 240倍.用PCR筛选文库中的3 000个单克隆,共获得9个阳性克隆,其中5个克隆含有长基因簇,4个含有短基因簇,并从中得到含有全长苯酚降解基因簇的克隆.利用以苯酚为唯一碳源的无机盐培养基,研究2个基因簇在大肠杆菌中的表达情况.结果显示,两个基因簇均表现出了苯酚降解能力,短簇的降酚能力要优于长簇.     

水稻染色体的显微分离与克隆

对水稻染色体识别、显微分离与切割、PCR体外扩增和微克隆进行了研究 .利用改进的显微切割装置 ,可以在 1 0 0×油镜下进行染色体分离与微切割 ,不仅解决了水稻染色体的识别和显微分离的困难 ,而且可使染色体特定区微切片段缩小到 7Mb级 ;利用 SUP-PCR可使 fg级水稻染色体 DNA扩增到 μg级 .电泳检测结果表明 ,扩增片断在 80～ 60 0 bp左右 ,经与 p UC1 9连接 ,转化大肠杆菌 DH5a,一次分离的单条水稻染色体可以获得 9×1 0 4克隆 ,一个 7Mb级片段可以获得 3× 1 0 4克隆 ,经分析 ,插入片段大小在 80～ 50 0 bp范围 .该方法为直接从水稻单条染色体或染色体特定区 DNA文库中筛选分子标记奠定了基础 .     

水稻S_5区候选克隆R2I19的筛选及序列信息学分析

以水稻广亲和品种Cpslo17为材料 ,构建了一个覆盖 9倍核基因组的cosmid文库 ,其平均插入片段约4 0kb。以与广亲和位点紧密联锁的单拷贝分子标记BAC2 3D12的R末端为探针 ,从cosmid文库中筛选得到一个阳性克隆R2I19(～ 32kb)。结合S5位点的高密度连锁图和物理图谱 ,初步确定为S5区候选克隆。对该克隆的 2个TAC亚克隆TRW15 10 (～ 15kb)及TRW15 17(～ 15kb)进行了初步的生物信息学分析 ,证实与已知的水稻基因组序列有很高的同源性 ,并显示其中可能含有与水稻育性相关的基因。     

利迪链霉菌A02细菌人工染色体基因组文库的构建

 利迪链霉菌A02是从京郊森林土壤中分离筛选出的植物真菌病害高效生防菌,其活性产物为安全高效广谱的抗真菌剂纳他霉素。为了克隆纳他霉素生物合成基因簇和调控其表达相关的功能基因,通过基因改良的方式进行A02的定向分子改造,提高纳他霉素的效价和产量,提取了利迪链霉菌A02的基因组DNA,用Hind III和Bam HI部分酶解后,回收了97~194kb和48.5~97kb大小的高分子量DNA,与质粒载体Copycontrol pCC1BAC连接,分别构建了含有800个和1500个克隆的两个BAC文库。从文库中随机挑选20个克隆,酶切检测平均插入片段分别为133kb和65kb,空载率小于1%,假定利迪链霉菌的基因组有8×10⁶kb,计算文库基因组覆盖率分别为12.28倍和11.25倍。因此,从文库筛选到目的片段的概率达99.99%以上。     

褐飞虱取食水稻幼苗cDNA文库的构建及筛选

以褐飞虱取食 3 2 h的水稻幼苗为材料构建了 c DNA文库 ,初始文库含 3 .2× 1 0 6 个克隆 ,重组率为 86%,取 1 .0× 1 0 6个克隆子扩增一次 ,收集到 80 m L滴度为 1 .2× 1 0 9pfu/ m L的扩增文库 .随机挑取 2 0个克隆以 T7和 M1 3 reverse为引物进行 PCR扩增 ,以鉴定插入片段的大小 ,结果发现多数片段的长度在 1～ 4.0Kb左右 ,少数片段在 4Kb以上 ,个别片段在 6Kb左右 .以在受褐飞虱取食的水稻幼苗中特异表达的 ESTBp Hi0 0 8为探针 ,筛选 c DNA文库 ,得到该基因的 c DNA全长     

活性污泥宏基因组Fosmid文库的构建

大插入片段宏基因组文库的构建是开发大片段目的基因及分析其结构与功能的基础.文中分别采用十六烷基三甲基溴化铵(CTAB)法、试剂盒及琼脂糖包埋法提取活性污泥宏基因组DNA,其中琼脂糖包埋法获得的DNA片段大于23kbp,利用此DNA成功构建了以pCC1FOS为载体的Fosmid文库,该文库含有5280个克隆,平均插入片段长度为35～40 kbp,共包含约200 Mbp的宏基因组DNA.从此文库中随机挑选200个克隆,利用活性筛选方法快速筛选到了1个含有淀粉酶的阳性克隆,表明活性污泥Fosmid文库可用于功能基因的活性筛选,具有开发新基因的潜力.     

Cupriavidus necator JMP134细菌人工染色体基因组文库的构建与鉴定

Cupriavidus necator JMP134(C.necator JMP134)可以降解60多种芳香族化合物,在环境污染治理方面具有良好的应用前景.该菌株基因组含有两个苯酚降解基因簇,克隆并研究其功能具有重要的理论和应用价值.以pIndigo-BAC 5为载体,构建了C.necator JMP134的细菌人工染...     

Physical location of the ricePi-5(t), Glh andRTSV genes by ISH of BAC clones

Mapping of low or single-copy sequences on plant chromosomes has proven difficult because of very low frequency of signal detection. Rice BAC library is being used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two biotin-labeled rice BAC clones closely linked to a rice blast resistance, green leafhopper resistance and tungro spherical virus resistance gene,Pi-5(t), Glh, RTSV, werein situ hybridized to rice chromosomes. They were located on the long arm and short arm of chromosome 4 with FL value of 40% and 100% respectively. The frequency of signal detection reached 46.8% and 59.2%. The signal location were consistent with the selective marker on rice saturated molecular map. The results demonstrated the advantages to locate BAC clones to chromosomes byin situ hybridization and will facilitate the rice low or single-copy gene location by using the BAC library. Supported by the National Natural Science Foundation of China and the Doctorate Vesting Point Foundation of the Education Department of the People's Republic of China Yan Huimin: born in 1964, Lecturer     

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and namedtms 1. Several AFLP markers which tightly linked to thetms 1 gene have been identified recently. In order to develop a detailed physical map of thetms1 gene-encompassing region and finally clone thetms1 gene, a bacterial artificial chromosome (BAC) library of rice 5460F (the fertile mutant line of TGMS rice 5460S) using a modified vector pECBAC1 has been constructed. The constructed 5460F BAC library consists of 16 896 clones with an average insert size of 119 kb, which represents about 4.7 times rice haploid genome equivalents. Neither chloroplast nor mitochondrial DNA was detected from the library. The library was screened with three single copy sequence amplified fragment length polymorphism (AFLP) markers which tightly linked totms1 gene as probes and eight positive clones were identified.     

Genetic and physical finemapping of the Sc locus conferring indica-japonica hybrid sterility in rice （Oryza sativa L.）

Hybrid sterility is a major hindrance to utilizing the heterosis in indica-japonica hybrids. To isolate a gene Sc conferring the hybrid sterility, the locus was mapped using molecular markers and an F2 population derived from a cross between near isogenic lines. A primary linkage analysis showed that Sc was linked closely with 4 markers on chromosome 3, on which the genetic distance between a marker RG227 and Sc was 0.07 cM. Chromosome walking with a rice TAC genomic library was carried out using RG227 as a starting probe, and a contig of ca. 320 kb covering the Sc locus was constructed. Two TAC clones, M45EI4 and M90J01 that might cover the Sc locus, were partially sequenced. By searching the rice sequence databases with sequences of the TACs and RG227 a japonica rice BAC sequence, OSJNBb0078P24 was identified. By comparing the TAC and BAC sequences, six new PCR-based markers were developed. With these markers the Sc locus was further mapped to a region of 46 kb. The results suggest that the BAC OSJNBb0078P24 and TAC M45EI4 contain the Sc gene. Six ORFs were predicted in the focused 46-kb region.     

Molecular cloning, chromosomal mapping and expression analysis of disease resistance gene homologues in rice (Oryza sativa L.)

Resistance-like sequences have been amplified from first strand cDNA and genomic DNA of rice by PCR using oligonucleotide primers designed from sequence motifs conserved between resistance genes of tobacco andArabidopsis thaliana. 3 PCR clones, designatedOsr1, Osr2 andOsr3 which were 98% identical in nucleotide sequence level, have been found to be significantly homologous to known plant resistance genes and all contained the conserved motifs of NBS-LRR type resistance genes, such as P-loop, kinase2a, kinase3a and transmembrane domain.Southern hybridization revealed that rice resistance gene hornologueswere organized as a cluster in the genome. RFLP mapping using a DH population derived from anindica/japonka cross (Zhaiyeqing 8/Jingxi 17) and an RFLP linkage map assigned two copies ofOsrl and one copy ofOsr3 to the distal position of chromosome 12 where a blast resistance QTL has been mapped previously. Northern blot analysis showed thatOsrl gene was constitutively transcribed in rice leaves, shoots and roots. Further study concerning isolation of full-length cDNAs would be conducive to elucidating the functions of these genes.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

A five-fold pig bacterial artificial chromosome library: a resource for positional cloning and physical mapping

A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind III or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480 pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10 single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.     

中国美利奴细毛羊BAC文库的质量评价

从文库的平均插入片段大小、文库的覆盖率、文库克隆的稳定性对中国美利奴细毛羊BAC文库进行了质量评价.统计结果表明,文库的平均插入片段大小约为133kb, 非重组克隆占3.2%,文库在理论上约有8倍绵羊基因组覆盖率,从该文库中找到任意单拷贝序列的概率为99.93%.连续多次传代培养实验证实文库克隆的遗传稳定性好.