基因文库的鉴定和应用

由于草菇是一种同宗结合的食用真菌，这给草菇的杂交育种带来了一定的困难。本文在建立草菇部分基因文库的基础上，对草菇的基因文库进行了鉴定，在草菇基因文库中任意抽取７２个克隆，利用专一的ＰＣＲ方法，测出在草菇基因文库中，草菇基因组ＤＮＡ的平均大小为１１５６个碱基对。在基因文库中任意选择５３个克隆，利用专一的ＰＣＲ进行ＤＮＡ扩增以及ｄｉｇ非同位素标记，用于和草菇基因组ＤＮＡ杂交。在测试的５３个克隆中有８％     

细菌基因组重复序列PCR技术及其应用

细菌中散在分布的DNA重复序列近年来不断被报道,基因外重复回文序列和肠细菌基因间共有重复序列是两个典型的原核细胞基因组散在重复序列。重复序列在染色体上的分布和拷贝数具种间特异性,用它们的互补序列作为引物,以细菌基因组DNA为模板进行PCR扩增反应,反应产物的琼脂糖电泳可以提供非常清晰的DNA指纹图谱,使用此图谱既可对各种微生物进行快速分型及鉴定,又可对它们进行DNA水平上的遗传多样性分析。细菌基因组重复序列PCR技术具有简捷、快速、结果稳定等特点,可对细菌进行分子标记,用于菌株分型、分类鉴定和亲缘关系等方面的研究。     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛（Budorcas taxicolor)属偶蹄目（Artiodactyla)、牛科（Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛(Budorcas taxicolor)属偶蹄目(Artiodactyla)、牛科(Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

鲫鱼Hind Ⅲ高重复DNA序列的分子克隆

基因组DNA高重复序列的研究有助于解释许多重要的生命现象 ,如基因调节、基因转座、基因进化等 ,还可以用于进行种群的遗传分析。鱼类的DNA高重复序列研究资料较少 ,曾在鲤科鱼类发现HindⅢ高重复序列家族。本研究用HindⅢ内切酶消化 ,从鲫鱼 (Carassiusauratusauratus)基因组DNA也克隆出一种独特的高重复序列。序列测定揭示该重复序列长度为 175bp ,在单倍体基因组的拷贝数为 1× 10 5。鲫鱼HindⅢ高重复序列与鲫鱼属 (Carassius)其它同类已知的高重复序列存在某种程度的变异 ,而与鲤科其它属的已知的HindⅢ高重复序列完全不同     

水稻重复序列RRD3在CHO和3T3细胞中的启动子活性

RRD3是通过变性 -复性动力学从水稻基因组中克隆到的一个中度重复序列 .将 RRD3作为启动子亚克隆到氯霉素乙酰基转移酶基因为报告基因的 p CAT载体上 ,转染 5种动物细胞 .结果表明在 CHO、3T3细胞中能够启动 CAT基因的表达 .利用核酸外切酶 和 PCR构建了 RRD3的系列缺失体 ,经转染后的 CAT活性分析表明 ,该序列中存在多个与哺乳动物细胞中启动基因表达有关的调控元件 .结合 RRD3在大肠杆菌和高等植物烟草中启动报告基因表达的活性 ,对重复序列与生物进化的关系进行了讨论 .     

巴氏蘑菇微卫星序列的分离及其对Co60辐射诱变菌株的鉴别

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。     

玉米特异DNA通过有性杂交导入小麦DH后代的分子证据

构建了玉米的随机基因组文库,筛选了近100个玉米基因组特异的重复DNA克隆,用它们作探针分别对2个来自小麦×玉米的小麦DH群体进行RFLP分析.结果发现,玉米的MR64克隆导入到2个小麦群体的各1株后代中,即在普通小麦DH系的18号株和波斯小麦DH系的15号株中检测到强杂交信号,这个结果首次从DNA水平上证明某些玉米特异的DNA序列,可通过受精作用以很低的频率转移到小麦DH后代的基因组中.测序分析证实,克隆MR64的插入片段长度为695Pb,A+T含量为58%,用多种酶切玉米基因组进行RFLP分析表明它是一个带1～3个主串联重复单位的散布重复序列.同时还对它的序列结构、甲基化图谱、拷贝数和在玉米染色体上的分布以及在其它小麦品种和禾本科种基因组中的序列同源性情况进行了详细的研究.     

辽东湾斑海豹(Phoca largha)线粒体D-loop区异质型研究初探

采用PCR技术和DNA克隆测序技术,随机测定了3头斑海豹mtDNA控制区(D-loop区)csn-3上、下游1000bp左右的序列,每头斑海豹任选14个克隆菌斑进行测序,结果所得序列均无重复,得到42个单倍型.结合GenBank已发表的斑海豹mtDNA控制区序列(Phoca largha,AM181031),通过Clusta1X1.83、MEGA3.1和FastPCRv3.6等生物信息学软件进行序列比对,发现我国珍稀保护动物斑海豹个体内线粒体DNA(mtDNA)的控制区CSB-3之后存在异质型现象,且存在数目不等的串联重复序列.从分子水平进行了不同类型重复序列变化规律的研究,初探了mtDNA控制区异质型在斑海豹中的存在情况.     

副溶血性弧菌重复序列-PCR分型研究

利用基因外重复回文序列-PCR(REP-PCR)和肠细菌基因间共有重复序列-PCR(ERIC-PCR)技术, 对副溶血性弧菌进行了分子分型研究和亲缘关系的探讨, 并使用Hunter和Gaston方法计算分辨力指数。结果显示40株副溶血性弧菌分离株均可扩增产生可重复的DNA指纹图谱, 并且不同菌株基因组DNA的扩增条带具有多态性。根据SPSS10.0软件得出的树状图结果, REP-PCR可以把40株菌分为21个型, 分辨力指数可达到0.953, 优势菌型为G1型; ERIC-PCR可将40株菌分成4个型, 分辨力指数为0.5。研究显示重复序列-PCR方法可以用于该菌分型分析, REP-PCR具有较好的分型能力。在两种PCR的DNA指纹图谱中, 血清型O1群与O3群主条带均非常相似, 表明它们之间亲缘关系密切。     

用分子生物学技术对草菇进行菌株鉴别

利用AP-PCR和RAPD技术对三个草菇菌株进行鉴别,其结果与用草菇菌株V34基因文库中的中等重复序列为探针进行限制性内切酶长度多态性分析(RFLP),及对编码核糖体5.8SrRNA的DNA(rDNA)进行PCR扩增后的产物进行限制性内切酶长度多态性分析(PCR-RFLP)的结果相一致。这一结果显示出用这四种方法对草菇菌株进行鉴别具有相似的效果。同时用这四种方法构建的分子生物学标记显示出这三个菌株中的两个菌株在遗传上有着较大程度的相似性。     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Characterization of swine short interspersed repetitive sequences

Swine genomic DNA segments containing repetitive sequences were isolated from a porcine genomic library using genomic DNA as a probe. Three fragments containing the repetitive sequences from two of the primary phage clones were subcloned for sequence analysis, which revealed six new PRE-1 repetitive families other than those reported earlier by Singer et al. (Nucleic Acids Research 15, 2780, 1987). The frequency of the repetitive sequences in the swine genome was estimated at 2 x 10(6) per diploid genome. Sequence analysis revealed similarities between these repetitive sequences and that of arginine-tRNA gene.     

Cloning of Y derived DNA sequences of bovine origin inEscherichia coli

We have constructed a partial library of Y chromosome derived DNA sequences of bovine origin inEscherichia coli. That, the recombinants arc Y derived and Y specific was ascertained by differential colony hybridization using male and female DNA probes. Out of 1000 recombinants analysed, 17 were found to be Y derived as well as Y specific and were of repetitive nature. Restriction analysis revealed that most of them had short DNA inserts.     

cDNA surveying of specific tissue expression of human chromosome 19 sequences.

cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.     

Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Two and five 1R chromosomes were microdissected from the metaphase spreads of rye ( Secale cereale L. ) root-rip cells with the aids of glass needles. The dissected chromosomes were amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 0.3 to 2.5 kb smear DNA fragments were obtained. After hybridized with DIG labeled probes, it was confirmed that the PCR products of the microdissected chromosomes were homologous with the rye genomic DNA, and derived from the 1R chromosome as well. Then, the second round PCR products from five chromosomes of 1R were microcloned to construct the plasmid library, including 220 000 clones. 172 randomly selected clones were evaluated ranged in size from 300 to 1 800 bp. Furthermore, the genomic dot hybridization results indicated that the library contained nearly 42% medium/high repetitive sequences and 58% low/single copy sequences, and its redundancy was very low. In this research, many aspects of the 1R chromosome microclone library exceeded or approached those of the previous reports in the literatures. Those are potential for construction of a high density genetic map of chromosome IR, from which some important genes can be tagged and isolated.     

Oligo-chip based detection of tick-borne bacteria

We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.