基于家系基因组测序对骡基因组结构多样性分析

为分析马(Equus caballus)和驴(Equus asinus)杂交后两套结构和功能完善的独立基因组在骡基因组中整合后的结构多样性，本研究以马属动物三成员家系的全基因组序列为研究对象，分别以纯血马基因组和驴基因组为参考，识别驴、马和骡的InDel、SNP和CNV；通过生物信息分析，分析骡的de novo SNP及其突变频率、识别骡特异性CNV，并进行功能分析。结果显示:1)驴、马和骡分别获得100.01、103.78和114.36 Gb 的高质量Illumina基因组测序数据。2)以纯血马基因组为参考，识别的InDels、SNPs和CNVs分别为402 533~2 110 786、5 012 403~23 819 055和2 126~3 761；以驴基因组为参考，识别的InDels、SNPs和CNVs分别为527 351~2 279 875、3 212 499~23 549 224和3 572~7 812。3)骡de novo SNPs分别为555和419，其突变频率为1.72×10^-7~2.21×10^-7。4)获得骡特异性CNVs分别为396和859，总长度分别为2.15和3.77 Mb。5)变异相关基因主要和机体的免疫及癌症过程相关。综上，骡基因组发生了高频率的de novo SNPs和特异性CNVs，这些结构变异可能对马和驴异种杂交不相容以及骡的遗传适应性具有重要意义，为进一步开展马和驴异种杂交的遗传基础及其分子机制的研究提供候选遗传位点。     

中药植物川牛膝基因组从头测序及分析

川牛膝是我国重要大宗药材,但近年来品质退化严重,品种真伪混杂。为深入研究川牛膝有效成分积累的分子基础,采用第二代测序技术利用Illumina Hi-seq 2000测序平台对川牛膝进行全基因组测序,使用AByss进行初步组装,得到一个包含大量基因组序列信息的数据集,并对其进行重复序列及编码序列注释。在该测序结果基础上,初步预测得到川牛膝体内甾体合成途径,并对鉴定川牛膝真伪的SCAR分子标记进行了初步定位,为研究川牛膝主要有效成分杯苋甾酮的合成途径,改良川牛膝品质提供了基础。     

A Bayesian networks approach for predicting protein-protein interactions from genomic data

We have developed an approach using Bayesian networks to predict protein-protein interactions genome-wide in yeast. Our method naturally weights and combines into reliable predictions genomic features only weakly associated with interaction (e.g., messenger RNAcoexpression, coessentiality, and colocalization). In addition to de novo predictions, it can integrate often noisy, experimental interaction data sets. We observe that at given levels of sensitivity, our predictions are more accurate than the existing high-throughput experimental data sets. We validate our predictions with TAP (tandem affinity purification) tagging experiments. Our analysis, which gives a comprehensive view of yeast interactions, is available at genecensus.org/intint.     

鸡瘦蛋白基因克隆中的若干问题研究

根据GenBank中鸡的LeptinmRNA序列(AccessionNo.AF012727)设计12对引物,用RT-PCR方法从不同品种(系)、不同时期及不同处理鸡的脂肪、肝脏和卵巢组织总RNA中没有扩增出鸡的Leptin基因片段;根据鸡的EST数据库中查到的一条鸡Leptin基因前体序列设计4对引物,用RT-PCR方法在包括卵巢在内的多个组织的cDNA中没能扩增出正确序列。为提高鸡Leptin基因的表达水平,通过给鸡注射胰岛素,利用RT-PCR方法对肝脏、卵巢和脂肪组织的总RNA进行扩增,没有得到目的序列。根据已发表的哺乳动物的Leptin基因序列设计兼并引物对鸡基因组DNA和脂肪、肝脏组织的cDNA进行PCR,结果没有特异性扩增条带,但在小鼠的基因组中可以获得稳定的扩增条带。用扩增长片段LATaq酶从鸡基因组DNA中也没有扩增出Leptin基因片段,而从小鼠的基因组DNA中,可扩增出小鼠Leptin基因片段;以小鼠LeptincDNA片段为探针,对鸡脂肪组织和肝脏组织来源的总RNA进行NorthernBlot分析,并未获得杂交信号;以猪的LeptincDNA片段为探针,对鸡基因组DNA进行SouthernBlot分析,并未获得特异性的结果。研究结果表明,在鸡的脂肪、肝脏和卵巢组织中不存在与小鼠Leptin基因同源性如此高的mRNA序列,在鸡的基因组中也不存在与小鼠、猪等哺乳动物Leptin基因序列同源性如此高的基因?     

Comparative Analysis of Genomes in Oryza sativa, O.officinalis, and O. meyeriana with C0t-1 DNA and Genomic DNA of Cultivated Rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosomes preparations of Oryza sativa, O. officinalis, and O. meyeriana with labeled probes of C0t-1 DNA and genomic DNA from the cultivated rice. The coverage percentage (%) and size (Mb) of C0t-1 DNA in O. sativa, O. officinalis, and O. meyeriana were 47.1 ±0.16, 38.61 ±0.13, 44.38±0.13, and 212.33± 1.21,269.42 ± 0.89, 532.56± 1.68 Mb, respectively. The coverage percentage and size of genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0, 93.6% and 634, 1 123 Mb, respectively, in which 365 and 591 Mb in O. officinalis and O. meyeriana were from O. sativa genomic DNA, but not from repetitive sequences of O. sativa, and the uncoverage genome size in O. officinalis and O. meyeriana were 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C0t-1 DNA in O. sativa, O. officinalis, and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during evolution. The repetitive sequences reduplication may be one of the important causes of the genome enlargement of O. officinalis and O. meyeriana, and O. officinalis genome enlarged more slowly when compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana were formed by reduplication, rearrangement, and gene selective loss during the evolution process.     

Recent segmental duplications in the human genome

Primate-specific segmental duplications are considered important in human disease and evolution. The inability to distinguish between allelic and duplication sequence overlap has hampered their characterization as well as assembly and annotation of our genome. We developed a method whereby each public sequence is analyzed at the clone level for overrepresentation within a whole-genome shotgun sequence. This test has the ability to detect duplications larger than 15 kilobases irrespective of copy number, location, or high sequence similarity. We mapped 169 large regions flanked by highly similar duplications. Twenty-four of these hot spots of genomic instability have been associated with genetic disease. Our analysis indicates a highly nonrandom chromosomal and genic distribution of recent segmental duplications, with a likely role in expanding protein diversity.     

鸭PRLR基因拷贝数的变异

本研究以台湾褐色菜鸭为试验动物,采用荧光定量PCR技术,使用2-ΔΔCt换算法对PRLR基因拷贝数进行定量,分析了其多态性与台湾褐色菜鸭生产性能的相关性.结果表明,PRLR和Ldh-B标准曲线的R2分别为0.991和0.990,扩增效率分别为111.68%和108.429%,斜率分别为-3.070和-3.135,PRLR和Ldh-B的扩增效率近似一致,可通过2-ΔΔCt方法对PRLR基因进行定量分析.在94羽台湾褐色菜鸭的样品中共检测到5种拷贝数(1、2、3、4、5)变异类型,拷贝数变异与台湾褐色菜鸭的蛋壳厚度和蛋形指数显著相关(P0.05),拷贝数为2的个体蛋壳厚度显著高于拷贝数为1和5的个体;拷贝数为3的个体蛋形指数显著低于拷贝数为1、2和4的个体;其余性状均无显著相关(P0.05).因此,PRLR基因拷贝变异区域可能影响蛋壳厚度和蛋形指数.     

Large-scale copy number polymorphism in the human genome

The extent to which large duplications and deletions contribute to human genetic variation and diversity is unknown. Here, we show that large-scale copy number polymorphisms (CNPs) (about 100 kilobases and greater) contribute substantially to genomic variation between normal humans. Representational oligonucleotide microarray analysis of 20 individuals revealed a total of 221 copy number differences representing 76 unique CNPs. On average, individuals differed by 11 CNPs, and the average length of a CNP interval was 465 kilobases. We observed copy number variation of 70 different genes within CNP intervals, including genes involved in neurological function, regulation of cell growth, regulation of metabolism, and several genes known to be associated with disease.     

蒺藜苜蓿叶绿体微卫星分布规律的研究

利用已公布的蒺藜苜蓿(Medicao truntula)叶绿体DNA(cpDNA)全序列测序结果,应用生物信息学软件对蒺藜苜蓿微卫星位点的分布情况进行了统计分析,结果表明:在已公布的124 033 bp的蒺藜苜蓿叶绿体基因组序列中,共有341个SSR序列,SSR的碱基总数达3 987 bp,约占整个基因组的3.2%;在所有SSR序列中,数量最多的是单碱基SSR,数量达到297个,占总数的87.1%,其次是二碱基重复序列,占12.6%,三碱基微卫星序列最少,仅有1个,大于三碱基的微卫星数为0;在单碱基重复中又以A和T重复为主,占单碱基重复总数的84.5%,二碱基重复也以AT和TA为主,占95.5%,单碱基中的纯粹重复类型占总数的65.3%。研究结果为该物种的分子标记的筛选提供了基础信息。     

利用水稻C0t-1 DNA和基因组DNA对栽培稻、药用野生稻和疣粒野生稻基因组的比较分析

【目的】研究中高度重复序列在稻属不同物种基因组进化中的作用。【方法】用栽培稻C0t-1 DNA和基因组DNA(gDNA)作为探针，分别对栽培稻、药用野生稻和疣粒野生稻进行荧光原位杂交(FISH)和比较基因组杂交(CGH)。【结果】C0t-1 DNA覆盖栽培稻、药用野生稻和疣粒野生稻基因组比例(%)和大小(Mb)分别为47.10±0.16，38.61±0.13，44.38±0.13和212.33±1.21，269.42±0.89以及532.56±1.68。栽培稻gDNA在药用野生稻和疣粒野生稻基因组中的覆盖率约为91.0%和93.6%，含量分别约为634 Mb和1123 Mb，各有365 Mb和591 Mb不属于源自栽培稻基因组的中高度重复序列，未被栽培稻gDNA所覆盖的部分，分别为64 Mb和78 Mb左右。此外，以C0t-1 DNA的组成为依据，对这3个种核型进行了同源性聚类。【结论】稻属中度和高度重复序列和功能基因一样，在不同种中也存在着高度同源性和保守性，并在进化过程中得以保存下来。药用野生稻和疣粒野生稻基因组增大的重要原因之一，可能是基因组中度和高度重复序列加倍的结果，药用野生稻这种序列扩增相对疣粒野生稻要缓和得多。另外，这两个野生种在长期进化过程中，由于存在加倍、重排和基因选择性丢失等现象，形成了具有自己种的特异性的基因组成分。     

小麦新品种川麦104的遗传构成分析

【目的】解析突破性高产小麦新品种川麦104的遗传构成,探讨双亲川麦42和川农16对其高产特性的贡献。【方法】利用已构建的遗传连锁图谱上的176个SSR和683个DArT标记对川麦104及其亲本进行分析,了解川麦104的遗传构成;根据已定位到的产量性状QTL,分析来源于双亲的染色体区段对川麦104产量相关性状的贡献。【结果】在川麦104的双亲具有差异的859个多态位点中（22个位点缺失）,有522个位点上的等位基因来源于川麦42,315个位点上的等位基因来源于川农16;川麦104更多地继承了川麦42的遗传成分（60.8%）;川麦104中来源于双亲的遗传位点在A、B和D基因组分布不同,来源于川麦42的等位位点在A、B和D基因组所占比例分别为55.00%、60.20%和67.27%;川麦104中来源于双亲的等位位点在21条染色体上的分布也不同,来源于川麦42的等位位点主要分布于3A、5A、7A、1B、5B、7B、3D、4D、5D和7D染色体上,来源于川农16的等位位点主要分布于4A、3B、4B、6B、1D、2D和6D染色体上。川麦104来源于双亲的染色体区段（遗传距离大于5 cM）共68个,总长度为3 089.6 cM;来源于川麦42和川农16的染色体区段分别为36和32个,来源于川麦42的染色体区段主要分布在3D、5D、7A、7B和7D染色体上,来源于川农16的染色体区段主要分布在3B、4B和6D染色体上;在A和D基因组川麦104来源于川麦42的染色体区段比川农16的多,B基因组中来源于川农16的染色体区段比川麦42的多。在1B、1D、2B、4A、4D、5A、5B、5D和7A染色体上,9个来源于川麦42的染色体区段以及5个来源于川农16的染色体区段富集了与产量性状相关的QTL,其中,在1BS和4A染色体上来源于川麦42的染色体区段携带增加穗粒数的QTL等位位点;在1D、2B和4A染色体上来源于川农16的染色体区段携带增加单位面积穗数的QTL等位位点;5B染色体上来源于川麦42的染色体区段和4A、4D染色体上来源于川农16的染色体区段均携带增加千粒重的QTL等位位点,这些QTL的聚合对川麦104的产量三因素有增效作用。【结论】小麦新品种川麦104的高穗粒数特性来源于川麦42,多穗数特性来源于川农16,其千粒重特性双亲均有贡献,表明双亲的正效产量性状QTL重组是川麦104的高产遗传基础。     

实时荧光定量PCR技术在检测外源基因拷贝数中的应用

通过对PCR法、Southern Blot法、IPCR法与实时荧光定量法4种转基因外源基因检测方法的比较认为:PCR扩增虽十分灵敏,但有时会出现假阳性扩增,因而对外源基因是否整合还需进行验证.Southren方法准确性高,特异性强,但存在费时、费力的缺点.同时实际操作中就需要较大量的植物材料来提取DNA,而转基因植物的愈伤组织在无菌条件下经过筛选、重新分化后,一般都比较细弱,不宜大量取样.IPCR法虽可以转座突变分离基因,但当转座子作为外源基因通过农杆菌介导等方法导入植物时,由于T DNA整合到染色体中引起插入突变并分离基因造成误差.实时定量PCR技术的特异性和高信噪比为转基因拷贝数定量提供了方便,实时定量PCR法,花费试剂少、节省劳力和时间,需要DNA样品量少、并不进行放射检测.同时对实时荧光定量PCR法计算进行了详细介绍.     

两种拼接方法在瓯江彩鲤转录组研究中的适用性比较

以瓯江彩鲤(Cyprinus carpio var.color)为研究对象,探讨了目前两种常用的转录组拼接方法在鲤的不同品种/品系转录组研究上的适用性。结果表明,在转录组拼接和注释方面,基于序列比对优先策略(Cufflinks软件)拼接的转录组序列平均长度为1 545 bp,注释77 601个转录本;基于从头拼接策略(Trinity软件)拼接的转录组序列平均长度为979 bp,注释转录本数为69 406个。在基因结构和选择性剪切预测方面,Cufflinks软件所拼接的转录组,每个基因平均含有3个转录本数目,而对于Trinity版本转录组,每个基因平均含有4个转录本(P0.001)。经比较发现Cufflinks版本转录组预测的基因结构较Trinity版本更加准确。因此,与从头拼接策略相比,序列比对优先策略所拼接的瓯江彩鲤转录本序列长度更长、注释的基因数目更多,且在基因结构和选择性剪切预测方面更为准确,适用于后续与功能基因表达相关的功能研究;相反,从头拼接策略拼接的转录组更易得到瓯江彩鲤的特异基因序列,对于后续需要利用瓯江彩鲤特异基因序列进行分子进化相关的研究非常重要。本文的研究结果为根据不同的实验目的合理地选用相应的转录组拼接方法提供了依据,也为其他鱼类转录组学研究提供了借鉴。     

Excess polymorphisms in genes for membrane proteins in Plasmodium falciparum

The detection of single-nucleotide polymorphisms in pathogenic microorganisms has normally been carried out by trial and error. Here we show that DNA hybridization with high-density oligonucleotide arrays provides rapid and convenient detection of single-nucleotide polymorphisms in Plasmodium falciparum, despite its exceptionally high adenine-thymine (AT) content (82%). A disproportionate number of polymorphisms are found in genes encoding proteins associated with the cell membrane. These genes are targets for only 22% of the oligonucleotide probes but account for 69% of the polymorphisms. Genetic variation is also enriched in subtelomeric regions, which account for 22% of the chromosome but 76% of the polymorphisms.     

Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation

Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.     

Frequency and Distribution of Microsatellites in the Genome of Filamentous Fungus, Neurospora crassa

A total of 38.0 Mb of publicly available DNA sequence in Neurospora crassa was researched for mono- to hexanucleotide simple sequence repeats (SSR or microsatellite) to determine the type, size and frequency. A total of 14 788 SSRs were observed in the whole genomic DNA sequence, about one every 2.57 kb, with the criteria of SSR length ＞15 bp and 80%matches. The most abundant microsatellite was trinucleotide repeat, the number was 4 729, followed by hexanucleotide and mononucleotide repeats, the numbers were 2 940 and 2 489 respectively, and the least abundance was dinucleotide repeat, only 691 were found. Among the 10 082 ORFs, 4 094 SSRs were harbored in 2 373 ORF (no intron) of the organism.One thousand and fifty six ORFs harbored only one SSR. Similar with other organisms, tri- and hexanucleotide repeats were predominant in ORFs, 54.1 and 48.8% oftri- and hexanucleotide repeats were distributed in ORF region. The density of these two motifs was overpresented in coding regions, because ORF region and coding region constitutes only 46 and 38.3% of genomic sequence, respectively. Upstream and downstream 300 bp of regulatory regions were high density regions of SSRs, particularly density of pentanucleotide SSR in upstream region was as high as five times of average density in genomic DNA, density of di- and tetranucleotide SSR was also more than two times of average density. The density of penta-, tetra-, di- and mononucleotide SSRs was relatively higher than average density. There were 47 SSRs in mitochondria 64 840 bp DNA sequence, their distribution is similar with genomic DNA sequence. These results suggested that SSRs were clustered in regulatory regions of genomic DNA.     

Gene amplification of c-myc and N-myc in small cell carcinoma of the lung

The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.     

基于定量PCR方法初步分析西藏开菲尔粒中细菌与酵母菌数量变化

运用实时荧光定量PCR方法分析西藏开菲尔粒中细菌与酵母菌的数量变化规律,需要有效提取其微生物总基因组DNA。本文通过同时添加溶菌酶与溶壁酶优化实验方案,成功获得西藏开菲尔粒中微生物的总基因组DNA。荧光定量PCR分析提取自培养1、4、8和12周的西藏开菲尔粒的微生物总基因组DNA,结果显示:4个时间点细菌16S rRNA与酵母菌26S rRNA基因拷贝数均分别约为每微升107拷贝与105拷贝,表明细菌与酵母菌的数量相对稳定。该结果为进一步研究西藏开菲尔粒中细菌与酵母菌间的共生关系奠定了一定的基础。