UniProt蛋白质数据库简介

UniProt(https://www.uniprot.org/)是国际知名蛋白质数据库,主要包括UniProtKB知识库、UniParc归档库和UniRef参考序列集三部分。UniProtKB知识库是UniProt的核心,除蛋白质序列数据外,还包括大量注释信息。UniProtKB知识库分Swiss-Prot和TrEMBL两个子库。Swiss-Prot子库中50多万条序列均由人工审阅和注释,而TrEMBL子库中1.4亿多条序列是由核酸序列数据库EMBL中的蛋白质编码序列翻译所得,并由计算机根据一定规则进行注释。UniParc归档库将存放于不同数据库中的同一个蛋白质归并到一个记录中以避免冗余,并赋予序列唯一性特定标识符。UniRef参考序列集按相似性程度将UniProtKB和UniParc中的序列分为UniRef100、UniRef90和UniRef50三个数据集。UniProt网站为用户提供了高效实用的高级检索系统和大量帮助文档。UniProt数据库每4周发布新版的同时也发布统计报表,用户可通过统计报表了解该数据库的数据量及更新情况、数据类别和物种分布等基本信息,查看常规注释信息、序列特征注释信息和数据库交叉链接等统计数据。UniProt是目前国际上序列数据最完整、注释信息最丰富的非冗余蛋白质序列数据库,自本世纪初创建以来,为生命科学领域提供了宝贵资源。     

家蚕胚胎发育时期的蛋白质变化及构造分析

采用蛋白质双向电泳技术及蛋白质氨基酸序分析技术,从蛋白质水平研究了家蚕胚胎发育时期的基因表达情况。结果表明,从家蚕临界期胚胎直到点青期胚胎的较长一段时间内,蛋白质的双向电泳图变化不大,匹配蛋白质斑点率达６３．０％,卵特异性蛋白质,３０Ｋ蛋白质的含量很大。     

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

家蚕雌性附腺及其Ng突变体蛋白质组双向电泳图谱分析

分别对家蚕(Bombyx mori．L)正常及Ng突变体雌蛾件附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析。用银染的方法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH4～8范围内,在分子量上主要集中在30～70kD区域：比较分析发现,有4种蛋白只在正常性附腺组织中特异表达,而有2种蛋白只在Ng突变体的组织中特异表达。另外约有29种蛋白在正常性附腺分泌部组织中的表达水平明显高于Ng突变,而约有15种蛋白在Ng突变体的分泌部组织中表达水平较高。这些差异蛋白质可能与Ng突变的形成和导致这种突变体的性附腺不能正常分泌粘性蛋白的性状有关。     

The amino acid sequence of two small ribosomal proteins from Bacillus stearothermophilus

The low-Mr proteins (tentatively called protein I and II) were purified from 2 M NaCl extracts of the Bacillus stearothermophilus ribosome. Their amino acid sequences have been determined from the peptides obtained by digestion with trypsin, chymotrypsin, and pepsin, and by cleavage with CNBr, using the micro-DABITC/PITC double-coupling method [FEBS Lett. (1978) 93, 205-214]. Protein I contains 56 residues and has an Mr of 6514. Protein II had 37 residues with an Mr of 4361. The amino acid sequence of protein I shows significant similarity to L32 from E. coli, whereas that of protein II is slightly, if at all, related to ribosomal protein L34 from E. coli.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

甜瓜STK类R基因同源序列的克隆与分析

以植物丝氨酸/苏氨酸蛋白激酶类( serine-threonine kinase,STK)抗病基因产物催化结构域I和Ⅸ的保守氨基酸序列( FGK/V/L/SVYK/RG,DY/IYSF/YGV/I/M)设计简并引物,对甜瓜(Cucumis melo L.)基因组DNA进行PCR扩增,得到大约500 bp的目的条带,通过重组质粒克隆并经PCR检测后得到12条不同的DNA序列,命名为tg1～tg12,其中tg2、tg5、tg9和tg12(Genbank登录号为JN646853 ～JN646856)可以编码完整的氨基酸序列.Blast分析结果显示:4条序列均具有ATP结合部位、底物结合部位和激酶结构域的活化环(A-loop)等,属于典型的蛋白激酶基因家族,可能是STK类R基因的同源序列片段;4条序列与蓖麻(Ricinus communisL.)的STK同源性均较高.氨基酸序列比对结果显示tg2、tg5、tg9和tg12均具有R基因的9个保守结构域,为STK类候选抗病基因类序列.分子系统树显示tg2、tg5、tg9和tg12与已知的R基因(Pto、Lr10和Lectin)在氨基酸水平上的相似性仅为33.5％ ～53.4％,且4个甜瓜同源序列的氨基酸相似性也较低,表明甜瓜RGAs标记可能具有较高的特异性.     

Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Daucus carota L.,and their partial amino acid sequences were determined.The homological analysis showed that the sequence from the 64 ku protein was highly homological to b -glucosidase,and that from the 55 ku protein had no significant homologue in GenBank.Using conservative sequence of animal IF proteins as primer,we cloned a cDNA fragment from Daucus carota L.Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves.Homological analysis revealed that it had moderate homology to a variety of a -helical proteins.Our results might shed more light on molecular characterization of IF existence in higher plant.