Structural analysis of a Lotus japonicus genome. I. Sequence features and mapping of fifty-six TAC clones which cover the 5.4 mb regions of the genome.

A total of 56 TAC clones with an average insert size of 100 kb were isolated from a TAC library of the Lotus japonicus genome based on the expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined according to the shot-gun based strategy. The total length of the sequenced regions is 5,473,195 bp. By comparison with the sequences in protein and EST databases and analysis with computer programs for gene modeling, a total of 605 potential protein-encoding genes with known or predicted functions, 69 gene segments, and 172 pseudogenes were identified. The average density of the genes assigned so far is 1 gene/8120 bp. Introns were identified in approximately 78% of the potential genes. There was an average of 3.8 introns per gene and the average length of the introns was 375 bp. DNA markers were generated based on the nucleotide sequences obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of L. japonicus Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

cDNA cloning and sequencing of Ca2+ calmodulin-dependent protein kinase IIα subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity, known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, se.) produces first mild ataxia followed by paralysis in 7-14 days in hens. DFP treatment also increases in vitro autophosphorylation of Ca²⁺ calmodulin-dependent protein kinase II (CaM kinase II) and the phosphorylation of several cytoslceletal proteins in the hen brain. To investigate whether increase in CaM kinase II activity is associated with increased expression of its mRNA, we cloned and sequenced CaM kinase II a subunit cDNA, and used it to study CaM kinase II expression in brain regions and spinal cord. Hen CaM kinase II subunit differs in 7 amino acids from that of rat CaM kinase II. Its mRNA occurs predominantly as a 6.7 kb message, which is very close to that of human CaM kinase II a subunit. Northern blot analysis showed a transient increase in CaM kinase II subunit mRNA in the cerebellum and spinal cord of DFP-treated chickens. The increase in CaM kinase II mRNA expression is consistent with the previously reported increase in its activity in brain and spinal cord, and its increased expression only in cerebellum and spinal cord, which are sensitive to the Wallerian-type degeneration characteristic of OPIDN, suggests the probable role of this enzyme in delayed neurotoxicity.     

MAR文库及其在真核基因组作图上的应用研究

基质结合区(matrix association regions,MAR) 是真核生物基因组中特有的一种参与基因表达调控和染色体动力学的顺式作用元件,也是真核染色体上的一种固有且各不相同的分子标记.通过体外(in vitro)或体内(in vivo)结合法可以得到与核基质特异性结合的MAR分子而构建MAR文库.体外MAR作为一种结构性的边界元件可用于染色体物理图谱的构建;而体内MAR作为某一组织中与核基质特异性结合的功能元件,则可用于研究已知基因的表达调控和染色体组织以及对新基因的分析.     

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.     

Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type I loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type I loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere- PRNP-(SODIL/AVP/OXT)-(BL42/GNAS1)-HCK-CSSM30 . The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.     

DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus,Scutellospora castanea

A technique has been developed to efficiently extract purified, restrictable genomic DNA from spores of different arbuscular mycorrhizal fungi in order to begin detailed investigations of the genome of the Glomales. The protocol yielded variable amounts of DNA depending on the fungal species; for Scutellospora castanea and Gigaspora rosea it reached values of 1.5–2 ng/spore. EcoRI digests of DNA from S. castanea were cloned into pUC18 and about 1000 recombinant DNA clones were obtained. Of those screened, 50 contained inserts of 500–7000 bp. Selected inserts detected DNA sequences from S. castanea spores or roots infected by this fungus, but not from nonmycorrhizal roots. This is the first report of a partial genomic library from an arbuscular mycorrhizal fungus.     

绿色木霉纤维素酶CBHII基因的分子克隆

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。     

Construction of a full bacterial artificial chromosome （BAC） library of Oryza sativa genome

We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 10^6 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.     

太原北齐徐显秀墓壁画真菌群落组成与菌害成因

【目的】确定壁画病害真菌群落组成,解析影响病菌发生的关键环境因子,为墓室壁画的科学保护提供依据。【方法】利用无菌解剖刀分别采集白色霉变与无明显霉变壁画样品;使用扫描电子显微镜(SEM)分析病害真菌微观形态特征;通过提取样品基因组总DNA、扩增真菌ITS区、构建克隆文库、测序和系统发生关系分析等技术研究壁画真菌群落组成与结构特点;结合温度与相对湿度监测,分析诱发壁画霉变的环境成因。【结果】霉变壁画表面有大量菌丝体,分生孢子大小为(1.5-2.0)μm×(1.0-1.5)μm。霉变壁画克隆文库序列分别与NCBI数据库中白色侧齿霉属(Engyodontium)和支顶孢属(Acremonium)真菌具有较高的相似度,白色侧齿霉菌(Engyodontium album)为优势病害菌(98.1%);无明显霉变壁画克隆文库序列分别与青霉属(Penicillium)、曲霉属(Aspergillus)、链格孢属(Alternaria)、假丝酵母属(Candida)、毛壳菌属(Chaetomium)和白色侧齿霉属真菌高度相似,无绒毛青霉菌(Penicillium laeve)为优势菌(77.4%);所有文库序列均属于子囊菌门(Ascomycota)。监测期墓道下部环境温度在-0.3-17.6°C之间波动,相对湿度长期在80%-100%之间变化。【结论】霉变与无明显霉变壁画中真菌群落组成差异较大;白色侧齿霉菌是引起墓道壁画霉变的主要病害菌;墓道下部相对湿度常年较高是诱发壁画霉变的关键环境因子;有必要开展壁画菌害区域的抢救性防护,并实施一定的环境控制措施以保护该考古遗址古代壁画。     

Abundance and Diversity of Methanotrophs and Propanotrophs in Soils above Yangxin Oil Reservoir,China

Methanotrophs have long been used as an important biological indicator for prospecting of oil and gas, while the indication of propanotrophs in hydrocarbon micro-seep systems is still poorly investigated. In this study, the abundance and diversity of the methanotrophic pmoA gene and the propanotrophic prmA gene as target genes were investigated in soils above Yangxin oil reservoir and Beiguan non-petroliferous area using molecular biological techniques. A total of 14 soil samples were collected at different depths (5, 20, 50, 100, 150, 200 and 250 cm) of two 2.5-m soil profiles located separately within the oil field and the non-petroliferous area for analysis of fluorescent quantitative real-time polymerase chain reaction (RT-PCR) (14 samples) and clone libraries (4 samples). The results demonstrated high presence of the propanotrophic prmA gene ranging from 7.68 × 10⁵ to 2.29 × 10⁷ copies/g dw (gene copies per gram soil of dry weight) in soil from the oil field relative to the non-petroliferous area for which the same measurements yielded results all below detection limit except for the 5-cm sample. On the other hand, oil field soil yielded much lower content of the methanotrophic pmoA gene (below detection limit to 5.6 × 10² copies/g dw) than the non-petroliferous area (1.14 × 10³ copies/g dw to 1.26 × 10⁵ copies/g dw) below 20-cm depth due to influence of biogenic methane, implying that propanotrophs may be better indicator bacteria for prospecting of oil and gas. Almost all pmoA clones of two 50-cm soil samples phylogenetically belonged to Gamma-Proteobacteria and the predominant pmoA OTUs were all uncultured bacteria. All prmA clones of two 5-cm soil samples were derived predominantly from Actinobacteridae (25.7%) and Alpha-Proteobacteria (74.3%), and all dominant prmA OTUs were also clustered with uncultured bacteria. Our results confirm that propanotrophs may be better indicator bacteria for prospecting of oil and gas and enrich the knowledge on diversity of methanotrophs and propanotrophs in the oil field and the non-petroliferous area.