Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.     

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased,and cost-effective method for the isolation of high molecular weight(23 kb) metagenomic DNA(260/280 ratio 1.8) with a good yield(55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination(eubacterial: human genomic DNA marker genes = 2~(27.9):1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16 S r RNA gene sequencing using 454 FLX+.Moreover, 16 S r RNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richnessand does not show microbial diversity biases, which is further confirmed by q PCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality,amount, user-friendliness, and cost effectiveness for its suitability toward usage for cultureindependent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.     

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.     

Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress （Thellungiella halophila）, a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

微生物全基因组鸟枪法测序

全基因组测序主要有二种策略,一种是分级鸟枪法测序,另一种是全基因组鸟枪法测序。微生物是一种十分重要的遗传资源,运用全基因组鸟枪法可以方便、快捷地完成其基因组的测序任务。本文对微生物全基因组鸟枪法测序中文库构建、插入片段的长短比例、反应投入量、拼接以及补洞等问题作了较细致的描述,有些步骤作了举例说明。 Abstract:Two strategies introduced for whole genome sequencing,one is clone by clone method,the other is whole genome shotgun sequencing,for microbes which are very important to us,whole genome shotgun sequencing method is very convenient.In this article we discussed the library construction、long-to-short-ratio of insert,、total number of reads should be sequenced、assembly and gap filling technologies of the whole microbial genome shotgun sequencing method while some examples presented.     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics.Isolation of genomic seg-ments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies.Herein,we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize.The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl ⅡMsp Ⅰ and ligation of digested fragments to the Bgl Ⅱ- and Msp Ⅰ-adaptors; 2) enrichment of a subset of Bgl Ⅱ/Msp Ⅰ fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking re-gions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions.Using this approach,we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-indueed mutant,and one such fragment,M196-FF,is found to contain a partial sequence of the DNA topoisomerase Ⅰ gene Topl.Moreover,the modified AFLP method including all restriction enzymes,adaptors and primers has been optimized in this study.The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.     

药用真菌子实体多糖SEC—HPLC图谱的相似度定量评价研究

目的：对灵芝属Ganoderma下4个种共7个菌株及灰树花Grifola ffondosa和云芝Trametes versicolor共9种子实体粗多糖进行了体积排阻HPLC色谱（SEC—HPLC）,并分析了相似度。方法：各样品水提物的HPLC图谱用平方欧氏距离法计算了相似度,用离差平方和法进行聚类分析。结果：灵芝属内各样品聚类距离为8,灰树花与云芝的聚类距离为16,它们与灵芝属的距离为25,说明同一灵芝属内的不同种及同种不同菌株所产生的多糖分子量分布近似度远高于来自树花菌属的灰树花或栓菌属的云芝。结论：此方法可量化描述不同多糖间SEC—HPLC的相似关系,有作为真菌多糖样品质量控制手段的潜在能力。     

灰树花多糖的分子修饰及其活性研究进展

灰树花是一种高蛋白低脂肪的食品,具有免疫调节、抗氧化和抗肿瘤等生物活性。其中,多糖是灰树花的主要活性成分之一。对多糖进行分子修饰是提高它原有的生物活性或增加新活性的重要途径之一。综述了灰树花多糖的分子修饰方法,以及修饰后灰树花多糖的生物活性及其构效关系的研究进展,以期为灰树花多糖的深入研究和开发利用提供参考。     

灰树花孔菌固体发酵基质抗氧化活性成分研究

选用燕麦、大豆、玉米、麸皮、大米、荞麦6种培养基质对灰树花孔菌进行固体发酵及抗氧化活性研究。结果表明：灰树花孔菌最佳抗氧化发酵基质为燕麦大豆培养基（燕麦16.25%,大豆83.75%）,最佳发酵时间为8d。灰树花孔菌燕麦大豆发酵基质抗氧化活性成分种类丰富,含量高。其中总黄酮物质40.86μg/g,总三萜5.94mg/g,多酚8.51mg/g,还原糖13.10mg/g,花色苷70.23μg/g,维生素C 24.57mg/g,维生素E 0.33mg/g,谷光甘肽（GSH）0.84g/g,SOD酶活性299.74U/g。     

微波辅助提取灰树花多糖工艺研究

采用提取时间、微波功率、液料比的单因素试验和正交试验法优化微波辅助提取灰树花多糖条件.结果表明,以净多糖得率为指标,影响微波辅助提取灰树花多糖的主次因素为:提取时间＞微波功率＞液料比,并且提取时间和微波功率的影响达到了极显著水平.灰树花多糖最佳提取工艺条件为:提取时间为10 min,微波功率为80%(全功率为800 W),液料比为25∶1.创立了一种用苯酚-硫酸法测定多糖时排除蛋白质干扰的方法.     

Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

Genomlc DNA polymorphlsms are very useful for tracing genetic traits end studying biological diversity among species. Here, we present a method we call the ＂diversity suppresslon-subtractlve hybridization array＂ for effectively profiling genomlc DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization （SSH） to establish e subtracted gDNA library, from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomlc DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphlc fragments of palrwlse comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array Is a highly effective platform for profiling genomlc DNA polymorphlsms and dendrograms.     

灰树花多糖对果蝇繁殖力及抗氧化作用的影响

实验采用超声辅助水提法提取灰树花多糖,以黑腹果蝇为实验对象,探究含0.2%,0.8%和1.6%浓度的灰树花多糖的培养基对果蝇繁殖力、体内脂褐素(LF)和过氧化氢酶(CAT)活性的影响。与对照组相比,各浓度组的灰树花多糖均能明显增强果蝇繁殖力,且对果蝇后代的雌雄比例产生一定影响;同时使果蝇体内LF含量明显下降,CAT活性显著升高。各浓度组间差异显著,尤以1.6%效果最佳。结果表明,灰树花多糖具有促进果蝇繁殖力和抗氧化的良好功效。     

灰树花多糖药理研究进展

真菌多糖的药理作用已得到广泛的研究与关注。作为一种独特的生物反应调节剂,灰树花多糖具有极大的开发利用前景。本文综述了国内外灰树花多糖药理研究的进展情况。     

用富集文库克隆人胰岛素基因组基因

通过构建可富集人胰岛素基因的λ噬菌体文库,克隆了人胰岛素基因组基因．首先从中国人血液白细胞中提取到人基因组ＤＮＡ,用ＥｃｏＲⅠ和ＢｇｌⅡ对基因组ＤＮＡ进行全酶切,经０．４％琼脂糖凝胶电泳,特异回收９．５ｋｂ左右的ＤＮＡ片段．将该片段与λＥＭＢＬ３／ＢａｍＨⅠ臂连接,构建成一个特殊的人基因组λ噬菌体文库（富集文库）,效价为２×１０４．同时采用ＰＣＲ方法及用引物Ⅰ：５′ＧＧＡＣＡＧＧＣＴＡＣＡＴＣＡＧＧＡＡＧＡＧＧ３′,引物Ⅱ：５′ＣＴＧＣＧＴＣＴＡＡＴＴＧＣＡＧＴＡＧＴＴＣ３′,从人基因组ＤＮＡ中扩增出一段含胰岛素基因的１．３６ｋｂＤＮＡ片段,做为放射性标记探针,对文库进行了噬菌斑原位杂交筛选,从１×１０４个噬菌斑中筛选到一个含人胰岛素基因组基因的阳性克隆,并进一步完成了亚克隆和该基因１７３２ｂｐＤＮＡ序列的测定．结果该基因的１７３２ｂｐＤＮＡ序列包括部分５′端和３′端与国外发表的人胰岛素α型等位基因的序列相同     

Suppression of D-galactosamine-induced liver injury by mushrooms in rats

Six species of edible mushroom were found to suppress D-galactosamine-induced enhancement of plasma alanine and aspartate aminotransferase activities when powdered mushrooms were added to the diet (5%) and fed to rats for 2 wk. Grifola frondosa exhibited the most potent effect in a dose-dependent manner. A significant effect was observed only from the water-soluble low-molecular-weight fraction of G. frondosa. The results indicate that several mushrooms possess a protective effect against liver injury induced by D-galactosamine.     

红脐鳞地衣基因组文库的构建(英文)

地衣是真菌和一种或多种光合微生物形成的稳定的共生联合体 ,既是先锋生物 ,又是敏感生物。环境的变化及生境的片断化 ,使得许多地衣种类处于濒危状态。保护珍稀濒危地衣物种的方法包括地衣体的移植 ,地衣中菌藻的分离培养及基因组文库的构建等。本研究用改进的CTAB方法提取基因组总DNA ,以Lamb daGEM 11为载体 ,构建了红脐鳞 (Rhizoplacachrysoleuca)的基因组文库 ,文库中同时含有该地衣共生菌与共生藻的DNA。该文库包含 8.5× 10 5个重组子 ,插入片段的平均大小为 19kb。文库的容量约为红脐鳞单倍体基因组的 10 0倍。该基因组文库的构建为保护稀有与濒危地衣物种提供了一个新的途径 ,并可进一步开展有关地衣的分子操作研究 ,如地衣冰核蛋白的异源表达等。