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.     

四川梅花鹿(CAG)n微卫星文库构建

应用Dynal磁珠-生物素标记的微卫星探针与四川梅花鹿基因组酶切片段杂交,捕获200～750 bp含有微卫星序列的DNA片段,连接pMD18-T载体,再转化到感受态细胞JM109中以构建文库.通过PCR方法从(CAG)n文库中筛选阳性克隆,从576个转化子中获得了234个阳性克隆,对其全部进行序列测定,其中73个含有微卫星序列.除获得的目的微卫星序列(CAG)n外,还观察到(AG)n、(AT)n的重复序列.本研究表明,经过优化的磁珠富集法能够稳定、高效地获得四川梅花鹿微卫星标记.     

云南松基因组微卫星富集文库的构建

采用磁珠富集法构建云南松微卫星富集文库。云南松基因组DNA经RsaⅠ酶切,与特定接头连接,再用接头特异引物进行PCR扩增。连接扩增产物与用生物素标记的(AG)12、(AT)12、(CG)12、(GT)12、(ACG)12、(ACT)12和(CCA)8探针杂交,通过链霉亲和素偶联的磁珠捕捉含接头和微卫星序列的片段并扩增,将获得的片段连接到pMD-19T载体上,转化至大肠杆菌JM109感受态细胞中,成功构建了云南松微卫星富集文库。通过PCR检测从文库中筛选阳性克隆,在383富集阳性菌落中获得阳性克隆257个,经测序分析,在获得的159条序列中,有143条含有SSR,其中完美型占65.73%,非完美型占23.78%,混合型占10.49%。结果表明,磁珠富集法构建云南松基因组微卫星文库高效、可行的,文库的构建为微卫星位点的分离、遗传多样性的分析等奠定基础。     

Dynal磁珠富集大熊猫微卫星标记

应用Dynal磁珠-生物素标记的微卫星探针与大熊猫基因组酶切片段杂交,捕获400—600bp含有微卫星序列的DNA片段,连接到pGEM-T载体中,构建富集微卫星序列的小片段插入文库。应用γ^32P标记的探针筛选文库,从2880个转化子中获得了260个阳性克隆。对54个序列进行了测序,并成功地设计了大熊猫微卫星引物37对。该方法能有效提高筛选微卫星标记的效率。     

藏羚羊(AC)_n,(AG)_n微卫星文库构建

藏羚羊Pantholops hodgsonii是我国珍稀的动物资源,开发其微卫星标记具有重要的科研与应用价值。本研究采用FIASCO法(Fast Isolation by AFLP Sequences Containing repeats)构建了藏羚羊的(AC)n和(AG)n微卫星富集文库。各文库随机挑取150个白色菌落,分别筛选到76个(AC)n和61个(AG)n阳性克隆;经测序得到含AC重复的微卫星序列37条和含AG重复的序列29条。研究共设计、合成微卫星引物47对,以扩增、电泳等方法进行筛选,最终获得15个可稳定扩增且具有多态的微卫星标记。筛选出的引物可用于评估藏羚羊的核DNA遗传多样性、遗传结构、种群动态以及构建藏羚羊遗传图谱等。     

岩原鲤微卫星富集文库的构建及微卫星分子标记的筛选

用磁珠富集法构建了岩原鲤Procypris rabaudi AC重复和GATA重复的微卫星富集文库.采用PCR方法分别以人工合成的oligoA和探针(AC)12或(GATA)6为引物筛选含有微卫星的阳性克隆.(AC)n 富集库和(GATA)n富集库的阳性克隆率分别为30%和7%左右.对40个AC重复的阳性克隆和30个GATA重复的阳性克隆测序,共获得61个微卫星序列,其中包含了一个三碱基重复(TGA)的微卫星序列.选择设计了19对AC重复和16对GATA重复的微卫星引物以及1对TGA重复的微卫星引物,通过PCR优化,共有20对引物能够产生稳定、清晰的目的产物带.为了检测获得的岩原鲤微卫星座位是否能用于近缘物种的研究,我们将20对岩原鲤微卫星引物用于中华倒刺鲃Spinibarbus sinensis基因组DNA PCR扩增,有60%的引物对能产生特异性的目的条带.本研究获得的20个岩原鲤微卫星座位可以用于岩原鲤及近缘物种的遗传多样性、种群遗传结构等的进一步研究.     

细梢小卷蛾微卫星富集文库的构建与分析

目的:构建细梢小卷蛾Rhyacionia leptotubula微卫星富集文库.方法:提取细梢小卷蛾基因组DNA,经限制性内切酶Rsa Ⅰ酶切,用(CT)10和(GT)10生物素探针与其杂交,利用磁珠富集含有微卫星的DNA序列,并对其进行PCR扩增,将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星富集文库:结果:对100个克隆进行随机测序,获得98个微卫星序列,其中具有5次及以上碱基重复次数的微卫星克隆占26%,最高碱基重复次数为33次,非完美型占12%,说明构建的细梢小卷蛾微卫星富集文库是一个高质量的文库.结论:该文库的建立为后续筛选具高多态性的微卫星标记引物研究细梢小卷蛾的种群遗传结构、迁移扩散规律等奠定了基础.     

牦牛基因组微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用链亲和素磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)12、(CCG)8、(CAG)8、(TTTC)8退火结合的含有接头和牦牛微卫星序列的单链限制性酶切片段,获得单链目的片段,经PCR扩增形成双链,然后克隆到pMD18-T载体上,转化至DH5α中,首次成功构建牦牛基因组微卫星富集文库。测序结果发现,阳性克隆率为77％(37／48),说明构建的牦牛基因组微卫星富集文库是一个高质量的文库。牦牛富集微卫星文库的建立和牦牛微卫星的筛选将为下一步进行牦牛基因组结构的分析、牦牛遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。     

云南松基因组微卫星富集文库的序列特征分析

目的:分析基因组微卫星富集文库的序列特征,探索云南松基因组微卫星的分布特征或变异规律。方法:采用磁珠富集法,从云南松基因组DNA的RsaⅠ酶切产物中富集微卫星片段,富集片段经洗脱分离、载体连接及转化等环节,对富集文库鉴定和测序,分析序列特征。结果:在测序获得的159条序列中,有143条序列含有重复次数不少于3次的微卫星序列,序列长度为135~1 138 bp,平均520 bp,克隆比例为89.94%。在这些序列中,以二核苷酸重复类型最多(56.64%),其次是三核苷酸,有51条(35.66%),此外,出现少量(7.70%)重复类型为4~6 bp的微卫星序列;各重复单元的碱基组成以探针及其互补序列为主,并出现少量的非探针类型;重复次数介于3~15,随着重复单元长度的增加而出现的频率降低。结论:分析了云南松基因组微卫星富集文库微卫星重复类型、重复单元碱基组成及其序列长度变异,为进一步分析云南松基因组微卫星的分布特征或变异规律奠定基础,进而为开发SSR引物提供基础信息。     

两种书虱微卫星富集文库的构建及比较

利用链霉亲和素与生物素之间的强亲和性原理,将链霉亲和素偶联的磁珠与微卫星探针(AC)12、(TC)12、(ATC)8、(ATG)8、(AAC)8、(ATAC)6及(GATA)6退火结合后,再亲和捕捉含接头和微卫星序列的单链书虱基因组DNA限制性酶切目的片段,经PCR扩增形成双链后进行克隆、建库。结果表明本研究成功构建了嗜卷书虱和嗜虫书虱共13个微卫星富集文库,包括6个嗜卷书虱文库,7个嗜虫书虱文库,其平均阳性克隆率为71.17%。经检测发现共得到两种书虱260个微卫星位点。这两种书虱微卫星富集文库的建立和高多态性微卫星位点的筛选将为嗜卷书虱和嗜虫书虱的种群遗传与进化、基因连锁图谱构建、分子系统发育研究等提供大量分子遗传标记,对其在实仓中的持续控制提供遗传学信息。     

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。     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

Probe-based negative selection for underrepresented phylotypes in large environmental clone libraries

Studies based on cloning and sequencing to investigate microbial diversity in a vast range of samples has become widespread in recent years. Results have revealed immense microbial diversity in many different environments, but also dominance of a few sequence types in the constructed clone libraries. Here we describe a method to enrich the clone libraries by avoiding sequencing of known, abundant sequence types, instead focusing on novel, rare ones. The protocol is based on gridding the PCR products from clone libraries on membranes and hybridisation of species-specific probes. Clones that do not give positive hybridisation results are sequenced. This method was used for fungal clone libraries from compost samples. Altogether 1536 clones were gridded and six probes used. From these clones, 59% hybridised with a probe, and therefore, only 41% of the clones were sequenced. In addition, 384 samples were sequenced to verify the hybridisation results. The numbers of false-negative (5.2%) and false-positive (3.9%) hybridisations were low. This method provides a mean of lowering the costs of sequencing projects and speeding up the process of characterising microbial diversity in environmental samples. The method is especially suitable for samples with a few dominating sequence types.     

Expressed sequence tags derived from brain tissue of Oreochromis niloticus

Expressed sequence tags (ESTs) were obtained from complementary DNA libraries derived from the brain of the cichlid fish, Oreochromis niloticus. Single-pass sequencing of 183 cDNA clones generated 294 ESTs; 57 of these clones (31%) were identified based on their similarity to sequences in GenBank.     

Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library

We constructed a high-efficiency expression library from Arabidopsis cDNA clones by introducing a poly (dC) stretch at the 5' end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high-efficiency expression library with antibodies raised against total proteins from Arabidopsis plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane-associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for Arabidopsis, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from Arabidopsis cells submitted to osmotic stress allows the selection of genes over- and under-expressed in stress conditions.     

Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by gamma-proteobacteria (53 to 64%), followed by beta-proteobacteria (18 to 21%) and alpha-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by alpha-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of delta-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.     

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.     

日粮中添加鱼油后肉牛瘤胃细菌区系的变化

目的：研究肉牛日粮中添加2%鱼油后瘤胃细菌区系的变化。方法：分别于添加鱼油前后取瘤胃液提取总DNA,根据细菌通用引物27F/1492R扩增16S rDNA基因序列,分别构建2个16S rDNA基因文库;从每个文库中各随机挑取384个克隆,对阳性克隆用HhaⅠ进行限制性片段长度多态性（RFLP）分析、聚类,取各RFLP类群中的代表克隆进行16S rDNA序列测定,构建系统发育树,研究细菌区系多样性的变化。结果：添加鱼油前、后,文库的RFLP图谱分别可以分成74和41个RFLP类群;添加鱼油前后的优势菌群均为噬纤维菌-屈挠杆菌-拟杆菌（CFB）和低（G＋C）含量的革兰阳性菌（LGCGPB）,但添加鱼油后文库中LGCGPB比例降低,而且未检测到纤维菌门细菌;有50%～60%的测定序列与GenBank数据库中的序列相似性高于97%,90%以上为未培养的细菌;添加鱼油后瘤胃细菌的多样性减少。结论：日粮添加鱼油后CFB比例增高但多样性下降,LGCGPB、纤维杆菌比例下降,这一结果为调控瘤胃发酵提供了依据。