柔嫩艾美尔球虫EST序列中SSR的获取及分析

对柔嫩艾美尔球虫EST—SSR进行生物信息学分析,共获取Eimeria tenella EST序列34074条,总长度为16．45Mb,小于12bpSSR的ESTs达7651条,从中获得SSR序列19576条、总长度为0．35Mb,EST—SSRs的频率是48．00％,平均相隔S40bp出现一个长度不小于12bp的SSR。在E．tenella的核苷酸重复基元中,2、3、4、5、6和7bp重复序列在基因组中出现的种类分别有11种472条、49种14710条、31种525条、13种25条、21种43条和15种400条,3碱基重复序列是最丰富的重复单元,占总数的75．14％。各种SSRs中富含G、C碱基的重复单元以GCA出现频率最多（28．63％）,次为AGC（17．59％）,GCT（8．76％）,TGC（7．62％）,CTG（7．15％）。     

Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins

While tandem mass spectrometry (MS/MS) is routinely used to identify proteins from complex mixtures, certain types of proteins present unique challenges for MS/MS analyses. The major wheat gluten proteins, gliadins and glutenins, are particularly difficult to distinguish by MS/MS. Each of these groups contains many individual proteins with similar sequences that include repetitive motifs rich in proline and glutamine. These proteins have few cleavable tryptic sites, often resulting in only one or two tryptic peptides that may not provide sufficient information for identification. Additionally, there are less than 14,000 complete protein sequences from wheat in the current NCBInr release. In this paper, MS/MS methods were optimized for the identification of the wheat gluten proteins. Chymotrypsin and thermolysin as well as trypsin were used to digest the proteins and the collision energy was adjusted to improve fragmentation of chymotryptic and thermolytic peptides. Specialized databases were constructed that included protein sequences derived from contigs from several assemblies of wheat expressed sequence tags (ESTs), including contigs assembled from ESTs of the cultivar under study. Two different search algorithms were used to interrogate the database and the results were analyzed and displayed using a commercially available software package (Scaffold). We examined the effect of protein database content and size on the false discovery rate. We found that as database size increased above 30,000 sequences there was a decrease in the number of proteins identified. Also, the type of decoy database influenced the number of proteins identified. Using three enzymes, two search algorithms and a specialized database allowed us to greatly increase the number of detected peptides and distinguish proteins within each gluten protein group.     

小桐子生殖组织基因表达谱

虽然表达序列标签(ESTs,mRNA片段序列)已广泛用于高效基因发现和补充基因组注释的工作,最近,与实时荧光定量反转录PCR(qRT-PCR)结合,它也开始应用于种系遗传学、转录谱及其蛋白组学方面.通过对油料木本植物小桐子(J.curcas)的生殖组织基因表达水平的分析,预期可能找到一些与油脂合成相关的基因.这些研究成...     

Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus

The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5′-untranslated region (UTR) of 17 bp, a 3′-UTR of 76 bp, and a stop codon TAA in 3′-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being challenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the immune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene discovery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expression studies in kelp grouper.     

Investigation on BRCA1 SNPs and its effects on mastitis in Chinese commercial cattle

The main objective of this study was to investigate whether the bovine breast cancer 1 (BRCA1) gene was associated with mastitis resistance in Chinese commercial cattle. A total of 51 SNPs were screened from public data resources and DNA sequencing. Three SNPs (c.5682G>C,c.26198C>T and c.46126G>T) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and created restriction site PCR (CRS-PCR) methods and 21 combinations of these SNPs were observed. The single SNP and their genetic effects on somatic cell score (SCS) were evaluated and a significant association with SCS was found in c.46126G>T. The mean SCS of individuals with genotype KK was significantly lower than those of genotypes KL and LL. The results of combined genotypes analysis of three SNPs showed that HHLLNN genotype with the highest SCS was easily for the mastitis susceptibility, whereas GGKKMM genotype with the lowest SCS was favorable for the mastitis resistance. The information provided in the present study will be very useful for improving mastitis resistance in dairy cattle by marker-assisted selection (MAS).     

花生EST数据库的分析、评价和应用前景

花生是我国重要的油料和经济作物。花生产业的发展对我国国民经济具有重要战略意义。随着分子生物学的发展,植物基因工程和功能基因组学的先进技术将有效推动花生种质创新和科技进步。分析了现有的花生EST数据,结合其他作物功能基因组学的最新研究进展,深入探讨了花生EST数据资源在基因克隆、分子标记开发及表达谱研究等方面的利用价值,并在分析花生EST数据库特点的基础上,展望了新一代测序技术在花生中的应用前景。为更好的利用花生EST数据库提供参考。     

Acanthamoeba castellanii: gene profile of encystation by ESTs analysis and KOG assignment

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.     

人肾上腺基因表达谱的建立及其功能的新认识

为深入理解人类肾上腺（AD）的功能,构建了正常人肾上腺cDNA文库,并利用大规模表达序列标签（ESTs）测序和生物信息学技术,研究显示参与基因/蛋白表达的基因类型表达最多,其次为能量代谢类.肾上腺中表达丰度最高的3个基因均为参与类固醇合成的酶类和蛋白.一些重要的基因首次显示在肾上腺表达,包括神经激素和神经肽,如促肾上腺皮质激素释放激素（CRH）,黑色素浓激素（MCH）,urocortin,可卡因和安非他明调节肽（CART）和垂体腺苷酸环化酶激活肽（PACAP）;许多重要介质的受体,如细胞因子、神经肽及神经递质受体;参与胆固醇代谢的基因,如LDL受体、HDL结合蛋白和胆固醇合成酶.研究结果表明在肾上腺表达丰度最高的基因与该器官的功能特异性有关,除类固醇激素外,许多神经肽、细胞因子在肾上腺产生,肾上腺与体内其他重要的系统间存在广泛的应答,而且在人肾上腺局部可能存在一个CRH-ACTH-皮质醇调节网络.