Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction （RAP.PCR） and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice, We believe that the findings described here will help to elucidate the molecular mechanism（s） underlying the developmental processes of rice leaf     

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.     

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.     

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.     

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.     

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 isadapted to harsh saline environments.To mine salt-tolerance genes from this species,we constructed an entry cDNA libraryfrom the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombination-assisted cDNA library construction method that is completely free of manipulations involving restriction enzymes andDNA ligase.This cDNA library construction procedure is significantly simplified and the quality of the cDNA library isimproved.This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for planttransformation and expression via recombination-assisted cloning.The library is plant transformation ready and is used totransform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

利用RACE技术扩增大豆抗病基因同源cDNA 5′末端序列

利用抗病基因保守序列筛选大豆cDNA文库,获得一抗病基因同源cDNA片段,命名为KR3-1。根据KR3-1设计两个基因特异引物（GSP 和 NGSP）,分别与通用引物(UPM)和巢式通用引物（NUP）共同扩增,成功地克隆到了该基因的5′末端序列。该扩增片段长447 bp,与已知序列重叠部分为129 bp。 Abstract:Based on part of a known partial cDNA sequence of a disease resistance gene homolog,KR3-1,obtained by screening a cDNA library from soybean,5′-RACE-PCR was carried out with gene specific primers and universal primers.After the nested PCR reaction,an amplified fragment of 447 bp in length which overlapped the known KR3-1 sequence by 129 bp was obtained subsequently.Thus,a 5′ cDNA end of KR3 was successfully cloned.     

蜘蛛基因组DNA Cosmid文库构建和拖丝蛋白基因的克隆

The genomic DNA Cosmid library was constructed from Nephila clavipes spider muscle using SuperCos 1 Cosmid as a vector.The title of library was >5×10 4 cfu/μg ligated DNA.On the basis of published sequence from a partial cDNA sequence of the 3′end of the dragline silk gene, we designed and synthesized 3 oligonucleotides.Oligonucleotides were labeled with non radioactive digoxigenin dUTP and detected with chemilluminescent substrate.56 positive recombinants were screen from the Cosmid library using DIG Oligo 2 as a probe.DNA dot hybridization using DIG Oligo 1 and DIG Oligo 3 as the probes, respectively, 3 positive signals were identified from 56 colonies.They were appeared the same pattern when DNA from the colones digested by restriction enzymes.The spider dragline silk gene was confirmed again by Southern blot hybridization.     

Identification of up—regulated genes in human uterine leiomyoma by suppression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas(Uls),suppression sub-tractive hybridization was used to construct an UL up-regulated library,which turned out to represent 88 genes.After two rounds of screening by reverse Northern analysis,twenty genes were proved to be up-regulated,including seventeen known genes and three genes with unknown function.All these genes were firstly associated with UL.Three genes with notable difference were selected for Northern confirmation.Our results proved the authenticity of the twenty genes.One gene named Phospholipase A2(PLA2) showed up-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obvious expression in prostate,testis,liver,heart and skeletal muscle.     

小麦抗病基因表达谱中的文库构建与筛选方法研究

以抗白粉病品系“百农 32 17×Mardler”BC5F4为材料 ,构建了白粉病菌诱导的普通cDNA文库和抑制消减杂交(SSH)cDNA文库。分别对两文库进行了一定规模的测序 ,获得普通cDNA文库不重复ESTs 387条和SSHcDNA文库ESTs 76 0条。将获得的ESTs与GenBank序列进行了BLASTn、BLASTx同源性分析。结果表明 :在普通文库中 ,一些参与光合作用与核糖体构成等的基因出现频率较高 ,而获得的抗病相关基因则较少。消减文库在构建方法、抗病相关基因的富集等方面具有明显的优越性 ,是目前抗病基因表达谱研究中的较好方法。利用高密度点阵膜杂交技术对两文库的筛选结果表明 ,该方法具有相对简便易操作、杂交膜可反复使用等优点 ;但也存在mRNA及同位素用量大等问题。经筛选 ,消减文库中有 5 4 1%的功能已知ESTs为抗病相关基因 ,被证明参与了小麦抗白粉病反应     

“电子”cDNA文库筛选指导基因的全长cDNA克隆

“电子”ｃＤＮＡ库筛选主要是指通过采用生物信息学的方法延伸表达序列标签（ＥＳＴ）序列,以获得基因的部分及至全长ｃＤＮＡ序列,避免或部分避免构建与筛选ｃＤＮＡ库等烦琐的实验室工作。该方法具体体现了ＥＳＴ数据库的迅速扩张已导致识别与克隆新基因的策略发生革命性的变化。ＥＳＴ序列ＺＡ７３为本实验室克隆到的可能参与辐射致气管上皮细胞恶性转化过程的基因片段,本研究采用“电子”ｃＤＮＡ库筛选的方法对其可能     

基于CRISPR-Cas9的筛选文库类型及应用

文库筛选技术广泛应用于生命科学研究各领域,加速了生物医药基础科研和临床实践的进展。本文对基于CRISPR-Cas9的文库类型和应用进行综述。CRISPR-Cas9文库包括敲除、活化和抑制文库。敲除文库通过Cas9/sgRNA靶向切割DNA序列,产生移码突变进行基因敲除。活化文库包括两种：一种是dCas9/sgRNA与转录活化蛋白质融合,例如dCas9-SAM,dCas9-SunTag和dCas9-VPR系统;另一种是dCas9与表观遗传修饰酶融合,例如dCas9-Tet1和dCas9-p300系统。CRISPR-Cas9抑制文库通过dCas9与表观遗传修饰蛋白质融合,抑制转录,例如dCas9-KRAB和dCas9-Dnmt3a系统。目前,CRISPR-Cas9文库广泛用于功能基因筛选、药物靶点和耐药靶点筛选、病毒靶点筛选和揭示信号通路,并在基因互作筛选及揭示顺式调节元件功能等方面初步展现其优势。CRISPR-Cas9文库优势在于其设计灵活、操作便捷、筛选高效。伴随基因编辑系统的研发,新的筛选文库靶向性和突变将更加精准,应用将更加拓展和深化。基于CRISPR-Cas9筛选文库不仅可以筛选病理和生理过程中的关键基因和非编码DNA,还可以揭示其发挥功能的分子机制,是剖析生命复杂调控网络的手术刀。     

温室黄瓜根结线虫发生地土壤微生物宏基因组文库的构建及其一个杀线虫蛋白酶基因的筛选

【目的】本研究旨在通过非培养手段构建和筛选宏基因组文库,以求找到新型的杀线虫蛋白酶基因。【方法】采用密度梯度离心法提取和纯化温室土壤微生物总DNA,经平末端、连接、包装、转染后,构建宏基因组Fosmid文库,同时,以脱脂奶为底物,以根结线虫为靶标,对文库进行功能初筛。【结果】该文库库容31008个克隆,平均插入片段36.5kb,包含1.13Gbp的微生物基因组信息,适合大规模的微生物功能基因筛选,通过功能初筛,筛选到1个含杀线虫蛋白酶基因的Fosmid克隆(pro12)。进一步构建和筛选出亚克隆(espro124a5),通过对基因结构进行了初步分析发现:espro124a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10(accession no.YP_756822at NCBI)的丝氨酸蛋白酶S15仅有45%的同源性,是一种新型的丝氨酸蛋白酶,有其保守的催化三元组:Asp469、His541和Ser348。【结论】密度梯度离心法提取到的DNA纯度高、片段长,完全能满足构建宏基因组Fosmid文库的要求;同时,构建的宏基因组Fosmid文库库容大,有利于我们从中筛选其他的微生物基因资源。     

牙菌斑培养菌群宏基因组文库构建及抗生素耐药基因筛选

[目的]构建牙菌斑培养菌群宏基因组文库,筛选牙菌斑生物膜中细菌的抗生素耐药基因.[方法]采集20例无龋健康人的集合牙菌斑并进行厌氧培养.提取牙菌斑培养菌群宏基因组构建Fosmid文库.用卡那霉素、四环素及氨苄西林对文库进行筛选,并对筛选到的抗性Fosmid克隆进行末端测序、亚克隆构建、亚克隆测序和序列分析.[结果]构建了牙菌斑培养菌群宏基因组Fosmid文库,插入片段长度在36-48 kb间约有15 120个克隆,插入片段长度小于36 kb的约有3 360个克隆.筛选获得一个氨基糖苷类双功能修饰酶AacA-AphD基因、一个核糖体保护蛋白型四环素耐药基因tet (M)及一个C家族β-内酰胺酶基因.[结论]证实了可以通过构建宏基因组文库的方法来筛选牙菌斑培养菌群中的抗生素耐药基因.     

噬菌体cDNA文库筛选方法的改良

cDNA文库的构建和简便、快速的筛选是获得全长基因的重要途径,基于PCR的筛库方法具有快捷、灵敏的特点。研究改进了基于PCR的噬菌体cDNA文库筛选方法,用液体分装的方法,替代了文库筛选的关键步骤——涂板分区,省去了噬菌体文库铺平板、浸染、培养、划块洗脱的操作过程,使筛库的工作量减少,进一步提高了筛选速度和获得阳性克隆的效率。     

用suc2信号肽捕获系统筛选小鼠胚胎cDNA文库基因

PCR扩增 1 1d小鼠胚胎cDNA文库插入片段 ,将 0 .5～ 2 0kb的扩增产物插入筛选载体的多克隆位点 ,转化suc2基因缺陷酵母宿主菌 .然后将约 1 0 5个酵母菌落接种于选择性平板上进行筛选 ,得到了 1 82个可在选择性培养基上生长的菌落 .PCR扩增显示 ,插入片段大小分布于 0 1～ 1 5kb之间 .对其中 1 4个阳性菌落的重组子进行序列测定 ,分别代表 6种不同的基因序列 ,与报告基因都有正确的读框内融合 .其中两种基因序列反复被筛到 ,分别命名为spt1、spt2 .spt1 [gi:2 772 876 6 ],可能以非编码RNA的身份参与蛋白质向细胞外分泌的过程 ,而spt2编码多个连续的赖氨酸 ,可能通过非经典途径介导蛋白质的分泌     

Construction of a cDNA library of Aphis gossypii Glover for use in RNAi

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site‐specific recombination of bacteriophage λ. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of 8.4 × 10⁶ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus‐induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi‐induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.