用RACE结合cDNA文库筛选的方法获取新的锌指蛋白基因

大多数有重要功能的蛋白质都含相应的由保守氨基酸顺序组成的功能结构域。本文首先根据蛋白质功能结构域保守氨基酸序列设计简并引物,用PCR方法扩增出基因EST序列,再利用改进的快速扩增cDNA末端（RACE）方法从cDNA文库中扩增出基因非同源部位,然后以非同源序列为探针,筛选cDNA文库。利用此方法成功地从人骨髓cDNA文库中克隆到几个编码锌指蛋白并代表原有EST的新的全长cDNA。这一策略也应适用于筛选编码具有其他序列保守性功能结构域蛋白的基因。 Abstract:Most of the important functionally proteins contain the corresponding function domains that consist of conserved amino acid sequences.The study provided a method to identify novel genes that encode proteins containing important functionally domains with conserved sequences.First,primers were designed according to the sequence of the cDNA library vector and the ESTs that have been obtained by reverse PCR and degenerate primers encoding Zinc finger domain.The cDNA library DNA was used as template for PCR amplification.The amplified fragment that contains nonhomologous sequences of the cDNA was inserted into pGEM-T easy vector.The fragment was recovered and used as a probe for screening the cDNA library.Several cDNAs with full length that encode proteins with Zinc finger domain and represent the original ESTs have been successfully cloned from a human bone marrow cDNA library.This strategy can also be used in screening genes that encode proteins containing differential function domains with conserved sequences.     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress （Thellungiella halophila）, a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

一种限制性cDNA文库的构建

利用本室创建的限制性显示技术RD-PCR,建立的cDNA文库,我们称之为限制性cDNA文库。该方法构建的文库因经过了限制性分组扩增,每组均含有特定的cDNA,因而大大加快了随后克隆的分离和鉴定的速度。 Abstract:A kind of cDNA library constructed according to Restriction Display PCR(RD-PCR) technology setup in our lab,which was called restriction cDNA library,was introduced in this paper.In the construction of cDNA library,cDNA was digested with restriction endonuclease,linked with special adaptor,amplified with PCR in groups.Each group of the restriction cDNA library contained special cDNAs.The method greatly reduced the repetitive frequency and accelerated the speed of identification.     

正常人体淋巴细胞cDNA文库的构建

应用GIBCOBRL建库试剂盒建立了正常人体淋巴细胞cDNA文库。取新鲜的正常人外周血,分离出淋巴细胞,进行体外培养,提取总RNA,纯化mRNA,并将其反转录成cDNA,与SalI和NotI接头连接后插入λZipLox载体,体外包装后转染到Y1090宿主菌中,进行滴度测试及文库扩增。构建的正常人淋巴细胞cDNA文库含2-6×106重组子,克隆效率为5×1012重组子/g cDNA,插入片段长度约为1~5kb。扩增后的文库浓度为3×107重组子/μl,将文库稀释到10-6时所产生的噬菌斑密度最为适宜。试验结果表明,该库符合标准,所构建的正常人淋巴细胞cDNA文库为进一步筛选目的基因、制作基因芯片等提供了有效的工具。 Abstract:A lymphocyte cDNA library of normal human was constructed in order to obtain specific gene and prepare lymphocyte gene chips to detect the relative genes between psychiatric diseases and immunity.The lymphocyte was abstracted from fresh normal human blood and cultured in vitro.Total RNA of lymphocyte was extracted from the cultured cells and then mRNA was extracted further.Moreover,single-strand cDNA and double-strand cDNA were synthesized in turn.The double-strand cDNAs were ligated to SalI and NotI adaptor,which were later ligated to arms of λZipLox.Ligated-cDNAs were packed in vitro,and then infected E.coli Y1090.Titering the phage and amplifying the library.The lymphocyte cDNA library consisted of 2-6×106 recombinants with the length of 1~5kb and the cloning efficiency was 5×1012 recombinants/g cDNA.The amplified library was 3×107recombinants/μl in concentration and the number of bacteriophage plagues was the most suitable in density after it was diluted to 10-6 in concentration.The constructed cDNA library of normal human lymphocyte would be helpful to further detecting target genes and preparing gene chips etc.     

Identification and characterization of a new member of serpin family- Hon-grES1 in rat epididymis

A full length cDNA named HongrES1 was isolated and cloned by screening rat epididymis cDNA library using a mouse EST as a probe and 5‘RACE followed. It contained 1590bp nucleotides and its predicted protein had 415 amino acid residues including a serpin (serine protease inhibitor) conserved domain. Tissue distribution pattern showed it was specifically expressed in adult rat epididymis; moreover, in situ hybridyza-tion indicated this gene was expressed in a limited region of the cauda epididymis near vas deference. Such kind of expression pattern sugested that HongrES1 had potential function in male reproduction.     

Cloning and characterization of a novel member of human β-1, 4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GAlT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1032 nucleotides (344 amino acids). In view of the homology to members of the galactosyltransferase gene family and especially the closest relationship to Gallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1, 4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.     

基于PC/Linux的核酸序列电子延伸系统的构建及其应用

新基因全长cDNA序列的获得常常是分子生物学工作者面临的难题。人类基因组计划及其相关计划的实施导致了大量表达序列标签(EST)的产生。利用一定的生物信息学算法,这些EST序列往往可用来对新基因片段进行延伸。采用Linux操作系统,利用Blast软件和Phrap软件以及EST数据库在微机上构建了EST序列的电子延伸系统,并对来自于人胎肝的11386条EST序列和511条插入片段全长cDNA序列进行了电子延伸,结果显示8373条EST序列和389条插入片段全长cDNA序列得到了程度不等的延伸,部分结果通过RACE实验得到证实。该套系统可高效地、规模化进行EST序列的延伸,可为通过实验获得新基因全长cDNA序列提供重要线索。 Abstract:Normally it is difficult to obtain full-length cDNA sequence of novel genes.More and more expressed sequence tags(ESTs) have been obtained since the start-up of human genome project.Powerful system is badly needed for data mining on these EST sequences.Based on a personal computer coupled with Linux operating system and EST database,the Blast software and Phrap software were used to construct a platform for in silico elongation of ESTs in our lab.The performance was tested using 11386 EST sequences and 511 partial-length cDNA sequences.Results demonstrated that 8373 EST and 389 cDNA sequence were elongated using this system.Thus the platform seems to be a fast way for full-length cDNA sequence cloning of new genes.     

略论棕榈科与新分出的省藤科的系统分类、演化、区系地理及主要的经济用途

棕榈科原省藤亚科因其子房壁及外果皮被倒生、螺旋状排列的鳞片所覆盖,而区别于其他亚科,因而独立分出成一新科--省藤科。作者讨论了棕榈科的祖先种可能在石炭纪时,自原始裸子植物开以顿目在分化、衍生出苏铁目祖先种的进化干上,于白垩纪时分化出的一个分支。在棕榈科的祖先种出现不久后,在其进化的分支上,于白垩纪后期又分化出一旁支,成为棕榈科的姊妹科--省藤科的祖先种。从两祖先种分别再分化、衍生出现今分布地球上该二科的属与种。两科、尤其前者是被子植物、尤其是单子叶植物中最原始的类群之一。作者还提出棕榈科象牙椰亚科与贝叶棕亚科是该科最原始或较原始的两类群;槟榔亚科和腊材榈亚科是较进化的两类群;而水椰亚科祖先种可能源于象牙椰亚科的祖先种,但又演化为该科最进化与特化的类群。省藤科省藤亚科略比鳞果榈亚科原始。作者讨论了两科为泛热带分布的科,指出两科的"现代分布区"在南北两半球热带地区,少数种还延伸分布到两半球暖亚热带、甚至达中亚热带地区,分布区边缘最北达日本中部、中国长江流域及黄河下游的南部,美国加利佛尼亚州与佛罗里达州和地中海北部;最南达智利中部和新西兰南部;而"现代分布中心"在热带与暖亚热带的亚洲,中、南美洲,大洋洲及非洲的东、南、西部;但分布区的"密集中心"则在热带亚洲、热带中及南美洲、南太平洋群岛及非洲东南部。作者还介绍了近50年我国南方引种驯化成功的两科植物近400种(见^*图谱),其中少数为耐寒的种类,有的种已引种到长江流域或更北的地区。引种的大部分种都有其重要的经济用途,包括:1. 食用,如淀粉和树液可制"西米"或制糖,酿酒、醋或作饮料;果或种子榨油,供食用或工业用;某些种的嫩芽作蔬菜,甚至种子代咖啡饮用;2. 药用,有消炎、止血、活血、驱虫、抗癌等用;3. 建筑、工艺与日用品,包括不少种的树干供建普通房子、桥梁、小船;少数种可提制工业用蜡;许多种的纤维制高级缆绳和编织品;还制工艺品与日用品等;4. 代表热带景观的园林工程、绿化及美化环境的观赏树和人行道树及建造园林景观生态类型的树种等。     

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose‐accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter‐relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone‐like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.     

The role of the microbiome in the neurobiology of social behaviour

Microbes colonise all multicellular life, and the gut microbiome has been shown to influence a range of host physiological and behavioural phenotypes. One of the most intriguing and least understood of these influences lies in the domain of the microbiome's interactions with host social behaviour, with new evidence revealing that the gut microbiome makes important contributions to animal sociality. However, little is known about the biological processes through which the microbiome might influence host social behaviour. Here, we synthesise evidence of the gut microbiome's interactions with various aspects of host sociality, including sociability, social cognition, social stress, and autism. We discuss evidence of microbial associations with the most likely physiological mediators of animal social interaction. These include the structure and function of regions of the ‘social' brain (the amygdala, the prefrontal cortex, and the hippocampus) and the regulation of ‘social’ signalling molecules (glucocorticoids including corticosterone and cortisol, sex hormones including testosterone, oestrogens, and progestogens, neuropeptide hormones such as oxytocin and arginine vasopressin, and monoamine neurotransmitters such as serotonin and dopamine). We also discuss microbiome‐associated host genetic and epigenetic processes relevant to social behaviour. We then review research on microbial interactions with olfaction in insects and mammals, which contribute to social signalling and communication. Following these discussions, we examine evidence of microbial associations with emotion and social behaviour in humans, focussing on psychobiotic studies, microbe–depression correlations, early human development, autism, and issues of statistical power, replication, and causality. We analyse how the putative physiological mediators of the microbiome–sociality connection may be investigated, and discuss issues relating to the interpretation of results. We also suggest that other candidate molecules should be studied, insofar as they exert effects on social behaviour and are known to interact with the microbiome. Finally, we consider different models of the sequence of microbial effects on host physiological development, and how these may contribute to host social behaviour.     

云南干热河谷植被与环境研究进展

特殊的地理位置和独特的地形地貌特征组合形成了典型的干热气候环境, 在云南省亚热带高原山地河谷下部发育了一类特有的植被类型, 即干热河谷植被。干热河谷植被具有非地带性和稀有性, 以及由土地利用变化为主的人为活动干扰导致的脆弱性。本文回顾了干热河谷植被的研究历史, 分别从干热河谷的植物群落学和植物区系学、干热河谷植被与土地的关系以及干热河谷植被保护与恢复三个方面进行了总结。植物群落与区系研究主要集中于群落分类、植被分类、群落特征、人为干扰影响、区系特征、性质和起源; 植被与土地关系研究侧重于土壤特性、土地利用/覆盖变化、土地退化及水土流失状况; 植被保护与恢复的热点在植被恢复目标、植被恢复功能区划、植被恢复引种及筛选及植被恢复效益评价研究。未来在这些区域应注重自然灾害及预防、水电工程建设对植被的影响及其响应等方面的研究, 深入开展大尺度植被时空格局的监测和动态服务功能分析。     

Physico-chemical environment in Tjeukemeer with special reference to speciation of algal nutrients

This paper attempts to generalize the major controlling factors and their impacts on the physico-chemical environment in Tjeukemeer in relation to the speciation of algal nutrients.Morphometry, man-made hydrology, climate, chemistry of drainage area and biology of the lake appear to control its physico-chemistry the most. Thus the shallow Tieukemeer is vulnerable to wind and therefore almost always completely mixed and often turbid owing to resuspended sediments. In normal summers the lake is oligohaline, hard, eutrophic and alkaline, and in winter it is hard, eutrophic, alkaline and humus-rich. The implications of these properties on the following physico-chemical components and their relevance for the speciation of algal nutrients are discussed: turbidity, colour, halinity, pH, alkalinity, salinity, conductivity, ionic composition, ionic balance and organic matter.Because of the relationships between the physico-chemical environment on the one hand and the speciation and bio-availability of nutrients on the other hand, special attention is given to the speciation of some major and minor elements as derived from ultra filtration experiments. In this context, the metal binding capacity of the organic matter (mainly fulvic acids) is also considered.     

人类口唇部形态变异的研究进展

口唇部是主要的面部形态区域之一,具有重要的生理功能。嘴唇保护口腔免受外部物质的渗透,维持口腔内部的湿度和温度,帮助咀嚼,同时也是外科美容整形手术中的主要部位之一,且在法医物证研究中唇纹也具有重要的鉴别价值。目前关于口唇部形态的研究主要包括以下几个方面：口唇部形态变异的性别、年龄、族群差异;影响口唇部形态变异的因素,如遗传基因、饮食结构、牙齿及颌骨形态和呼吸方式等;口唇部形态研究在医学、个人身份识别及法医刑侦领域的相关应用等。国内口唇部形态研究相对较少,尤其缺乏较为系统的口唇部形态生长发育及变异研究。本文通过梳理和归纳国内外相关文献的研究数据和结论,对口唇部形态相关研究作简要概述,并对国内口唇部生长发育及形态变异研究作了简要回顾和展望。     

How Romanowsky stains work and why they remain valuable — including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme

Abstract

An introduction to the nomenclature and concept of “Romanowsky stains” is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of “the color purple.” Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of “the color purple,” the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a “universal” generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye–basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.     

IPBES工作效率和科学职能的有效性分析

IPBES第一轮工作方案受到国际社会的广泛关注,奠定了其作为第一个生物多样性领域政府间机制的重要地位.IPBES自成立以来相继发布了系列评估报告和决策者摘要,引起了国际社会广泛关注,因此,其公平性、公正性、科学性和透明性始终为各界关注的焦点,直接决定着IPBES评估报告的可信度和未来的发展.为有效提升工作效率,提高其成...     

中国昆虫染色体研究现状与展望

简要叙述了中国昆虫染色体研究的现状,包括研究涉及的昆虫类群、核型分析结果、研究方法和手段、染色体有丝分裂、减数分裂、染色体形态变异、结构变异和数量变异等。我国学者对昆虫染色体研究从20世纪30年代开始,迄今已对蜉蝣目、蜚蠊目、直翅目、半翅目、同翅目、鞘翅目、鳞翅目、双翅目、蚤目和膜翅目等10目481种昆虫的核型进行了研究,主要集中在蝗虫、蝽类、蚜虫、蚕类、果蝇、摇蚊及实蝇等。在染色体行为方面的研究主要有：蚕类和果蝇等有丝分裂;蜚蠊类、蝗类、蝽类和蚕类的减数分裂及性别决定机制;部分昆虫的联会复合体分析。染色体结构变异的研究主要集中在果蝇和蚊类昆虫的唾腺染色体;果蝇的B染色体;蚕类和蚊类昆虫染色体的缺失、易位和倒位等变异;蚕蛾类的数量变异。研究结果多应用于昆虫系统分类和进化的探讨,揭示昆虫遗传与变异规律。通过与国外研究成果对比,提出昆虫染色体研究的必要性,并对我国未来昆虫染色体研究进行了展望。