基于亲权鉴定的千岛湖社鼠家群遗传结构与亲缘关系特征研究

于2009年7月至2010年11月,对浙江千岛湖两个岛屿上的社鼠(Niviventer confucianus)种群进行标志重捕,并采用8个高多态性的微卫星位点,对两个岛屿的社鼠种群进行家群分析和亲权鉴定,探讨了社鼠家群的亲缘关系特征。结果显示,8个微卫星位点能可靠地对两个岛屿社鼠种群进行亲权鉴定,A岛已确定亲缘关系的71只社鼠分为12个家群,家群中的个体数最多达到19个,B岛已确定的49只社鼠个体共分为11个家群,家群中的个体数最多达到14个。家群内部成员之间的亲缘关系表现为配对繁殖的个体对间亲缘系数最小,揭示了社鼠倾向于选择亲缘关系较远的异性作为配偶。家群中雄性后代个体之间与雌性个体之间的亲缘关系相比,两岛表现情况相反,该结果暗示两岛屿上社鼠扩散行为可能有所不同。通过计算与同一雄性(或同一雌性)交配的个体间的亲缘系数,发现两个岛屿上的社鼠在与不同异性交配时也存在选择性,即避免选择亲缘关系较近的异性作为混交的对象。     

人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定

采用聚合酶链反应(PCR)从人脑cDNA文库中扩增出600bp的髓鞘碱性蛋白(MBP)cDNA片段,与载体pGEM-3Zf(+)平端连接.重组质粒DNA转化宿主菌JM109,在含X-gal和IPTG的平板上直接筛选阳性克隆.限制性内切酶分析和成套引物扩增鉴定证明,该克隆含有7个外显子的21.5kD人脑MBP全长编码序列.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

银合欢基因文库的构建和种子贮藏蛋白基因的分离

本文以λEMBL_3为载体,构建了银合欢(Leucaena leucocephala)的基因文库,所得重组子为3.5×10~6pfu。以大豆种子贮藏蛋白α′亚基基因为探针,从基因文库中分离得到了4个阳性克隆,并初步绘制了其中3个重组子的物理图谱。结果表明在这3个重组子的基因内部有一致的酶切位点。亚克隆的部分核苷酸序列与 GenBank 中的基因序列比较,表明与大豆种子贮藏蛋白α′亚基基因高度同源。     

DPHL:A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.     

Isolation and characterization of microsatellite loci in a polyploid alpine herb,Callianthemum miyabeanum (Ranunculaceae)

• Premise of the study: Nuclear microsatellite primers were developed to analyze the clonal diversity and population genetic structure of the endemic polyploid herb Callianthemum miyabeanum. • Methods and Results: Using a protocol for constructing microsatellite-enriched libraries, 15 primer sets were developed for use in C. miyabeanum. The number of alleles found ranged from five to 22. The estimated range of expected heterozygosities was 0.574 to 0.907, and the Shannon–Weiner diversity index ranged from 1.061 to 2.733. Cross-amplification of all loci was also successful in the closely related endemic species C. kirigishiense and C. hondoense. • Conclusions: The development of these microsatellite loci will facilitate a deeper understanding of the genetic diversity, mode of reproduction, and population structure of not only C. miyabeanum, but also the other Callianthemum species endemic to Japan.     

Isolation and characterization of microsatellite DNA loci in sea bass,Lates calcarifer Bloch

Polymerase chain reaction (PCR)‐based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.