基因表达系列分析法(SAGE)的改进及其在植物功能基因组研究中的应用

基因表达系列分析方法(SAGE)是一种新的基因表达分析方法,与基因芯片技术一样具有高通量的特点,可测定特定组织的基因表达水平,在全基因组水平上同时定量检测数万个基因表达模式;可在未知目的基因的前提下,分析来自一个细胞的全部转录本信息;对已知或未知基因表达进行定性和定量分析.目前,虽然在疾病、发育、细胞凋亡、药物筛选等多个领域已有利用SAGE方法进行的研究,但该方法在植物功能基因组研究中的应用相对较少.本文主要综述了该方法在RNA用量、PCR循环次数、SAGE效能和可靠性、标签长度和未知标签分析等方面的改进及其在植物中构建SAGE文库、筛选新基因、基因表达图谱分析等方面的应用,从而为其在植物功能基因组研究中的进一步应用提供理论参考.     

Convenient genotyping of six myostatin mutations causing double-muscling in cattle using a multiplex oligonucleotide ligation assay

We herein describe a procedure that allows for simultaneous genotyping of six loss-of-function mutations in the bovine myostatin gene associated with the double-muscling phenotype. The proposed method relies on a multiplex oligonucleotide ligation assay and detection of the fluorescently labelled products using automatic sequencers.     

Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

Study on fibroin heavy chain of the silkwormBombyx mori by fluorescencein situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized theFib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified thatFib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

桃ERF转录因子家族生物信息学分析及 芽萌发相关基因筛选

以中油四号油桃(Prunus persica var. nectarina)为研究对象, 利用MEGA 6.0、MEME、GSDS和DNAMAN 6.0等软件对桃ERF家族数据进行生物信息学分析, 鉴定得到102个ERF转录因子家族基因, 并通过构建系统进化树将这102个基因分为10个子家族(I-X)。基因结构分析表明, 有81个基因不含内含子, 20个基因含有1个内含子, 有1个基因与其它成员差异较大, 含有5个内含子。保守元件分析表明, ERF家族包含20个保守元件, 其中Motif 1、Motif 2和Motif 4都属于AP2/ERF结构域, 同一个保守元件主要出现在同一个子家族中, 并且大部分保守元件的功能未知。VIII子家族基因的荧光定量PCR分析表明, 在桃叶芽处于不同的发育状态时, PpeERF068的表达量存在较大差异, 光照培养箱中培养的桃芽在萌发过程中各时期表达量变化趋势进一步表明该基因可能与叶芽萌发有关, 将其命名为PpeEBB1。该研究为进一步揭示PpeEBB1的分子机制奠定了基础, 并为桃树的栽培管理和熟期调控了提供理论指导。     

Testing hypotheses driving genetic structure in the cooperatively breeding Brown‐headed Nuthatch Sitta pusilla

Habitat fragmentation has often been implicated in the decline of many species. For habitat specialists and/or sedentary species, loss of habitat can result in population isolation and lead to negative genetic effects. However, factors other than fragmentation can often be important and also need to be considered when assessing the genetic structure of a species. We genotyped individuals from 13 populations of the cooperatively breeding Brown‐headed Nuthatch Sitta pusilla in Florida to test three alternative hypotheses regarding the effects that habitat fragmentation might have on genetic structure. A map of potential habitat developed from recent satellite imagery suggested that Brown‐headed Nuthatch populations in southern Florida occupied smaller and more isolated habitat patches (i.e. were more fragmented) than populations in northern Florida. We also genotyped individuals from a small, isolated Brown‐headed Nuthatch population on Grand Bahama Island. We found that populations associated with more fragmented habitat in southern Florida had lower allelic richness than populations in northern Florida (P = 0.02), although there were no differences in heterozygosity. Although pairwise estimates of F_ST were low overall, values among southern populations were generally higher than northern populations. Population assignment tests identified K = 3 clusters corresponding to a northern cluster, a southern cluster and a unique population in southeast Florida; using sampling localities as prior information revealed K = 7 clusters, with greater structure only among southern Florida populations. The Bahamas population showed moderate to high differentiation compared with Florida populations. Overall, our results suggest that fragmentation could affect gene flow in Brown‐headed Nuthatch populations and is likely to become more pronounced over time.     

Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified (“dark peptidome”) by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC.     

栗背短脚鹎的种群分化与遗传多样性

栗背短脚鹎(Hemixos castanonotus)是我国南方山区常见的杂食性鸟类。为探明其遗传多样性及分化现状,采用线粒体Cyt b基因和7个核基因非编码区片段作为分子标记,对分布于广东、广西、海南、贵州和江西五省(自治区)的栗背短脚鹎11个地理种群进行了遗传分化及遗传多样性研究。基于所获得的Cyt b基因866 bp和7个核基因内含子序列6 808 bp进行分析。结果显示,在Cyt b基因中,共检测到37个单倍型,共享单倍型占单倍型总数的35.6%,推测这些共享单倍型可能属于祖先单倍型。分子方差分析结果显示,遗传变异主要来源于种群内部(79.77%)。Tajima’s D和Fu’s Fs中性检验分析结果均支持栗背短脚鹎种群可能曾经历过种群扩张现象。基于7个核基因内含子联合序列的贝叶斯天际线(BSP)分析,推断其种群在大约5.3 ~ 3.7百万年前(Mya)和约0.7 ~ 0.3百万年前(Mya)发生过扩张。基于Cyt b基因的贝叶斯系统发育分析,11个地理种群共分为两支,一支为海南猴猕岭地理种群,属指名亚种(H. c. castanonotus),其他10个地理种群聚为另一支,属H. c. canipennis亚种,并且后者尚未形成显著的地理结构,单倍型网络图分析也获得相似的结果。本研究所用分子数据基本支持两个亚种的分化,对于存在争议的广西南部分布的指名亚种,其分子数据与形态学亚种归属不一致,有待更深入研究。     

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.