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1.
水稻插入突变库构建研究进展   总被引:6,自引:0,他引:6  
水稻是单子叶植物基因组研究的一种模式植物 ,其全基因组测序已经完成 ,在此基础上开展功能基因组的研究。水稻插入突变体库的建立是功能基因组研究的一个重要内容 ,在此基础上也能进行正向遗传学及反向遗传学的研究。水稻插入突变体库构建的方法有T DNA插入突变、Ac Ds系统插入突变、Tos1 7插入突变。分别介绍三种方法的原理及其在水稻突变体库构建中的应用和研究进展。  相似文献   

2.
戚飞  林硕  樊启昶 《遗传学报》2004,31(7):750-757
用于大规模基因突变与筛选的主要策略有化学诱变、插入突变、基因诱捕。插入突变是一种通过外源DNA整合的方式来获得突变体,并克隆得到对应突变基因的方法。运用反转录病毒介导的插入突变技术,在脊椎动物斑马鱼中已经获得了许多影响胚胎发育和细胞生长过程的突变体,并找到了对应的基因。基因诱捕技术也被运用于反转录病毒载体的构建。这套系统的建立使斑马鱼成为第一个有可能达到基因饱和突变和筛选的脊椎动物。  相似文献   

3.
We have constructed a plasmid useful for insertional mutagenesis inStreptococcus mutans.The molecule, pSU20Erm, is based on a derivative of pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates inEscherichia coli,does not replicate inS. mutans,contains an erythromycin-resistance marker which can be selected inE. colior the streptococci, contains a multiple cloning site with few restriction sites in the remainder of the molecule, and can be screened on X-Gal-containing medium for the presence of insertions into the multiple cloning site. We have used the plasmid to construct a library ofS. mutansDNA inE. coliand show that the clones can be reintegrated into theS. mutanschromosome via homologous recombination, thereby interrupting native genes. The plasmid has been used to clone part of a homologue of theE. coli drpAgene, encoding a global regulatory element for RNA synthesis. Further, we have identified an element closely linked todrpAinS. mutanswith high homology to IS861.  相似文献   

4.
稻瘟病菌T-DNA插入方法优化及其突变体分析   总被引:10,自引:0,他引:10  
优化了农杆菌介导转化稻瘟病菌获得T-DNA插入突变的条件,包括选择转化子的潮霉素B用量,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比,不同转化阶段培养基的选择等。转化1×106个孢子平均可获得约500个左右的转化子,PCR和TAILPCR检测表明约85%转化子中含T-DNA插入。对1520个突变体进行形态变异观察,发现菌落颜色突变的有15个;随机取58个突变体进行比较,发现产孢量减少的4个,孢子萌发率降低的8个,附着胞形成率降低的9个;还获得对水稻品种C101LAC(Pi-1)和751127(Pi-9)致病的突变体,为进一步克隆相应的无毒基因奠定了基础。  相似文献   

5.
新一代测序技术(Next-generation sequencing,NGS)在阐明复杂和高度重复的基因组结构,DNA序列与基因组结构变异同重要农艺性状之间的关系等方面具有重要作用。从NGS系统的开发与作物基因组测序,NGS与转录组分析,NGS与全基因组关联图谱,及SNPs开发与预测育种等方面,综述了NGS技术在作物基因组研究中的应用,可为作物基因组研究提供理论基础。  相似文献   

6.
转座子Tn917诱变的炭疽杆菌芽孢形成缺陷株的筛选   总被引:1,自引:0,他引:1  
目的:诱导转座子Tn917随机插入炭疽杆菌染色体,产生在不同位点突变的突变体库,从中筛选芽孢形成缺陷型突变株。方法:用含转座子Tn917的质粒pLTV3转化炭疽杆菌,以低浓度红霉素诱导转座因发生转座,产生大量的突变株。进而用氯化三苯基四氮唑染色法和复红美蓝染色法从突变体库中筛选芽孢形成缺陷株;用Southern杂交法对芽孢形成缺陷株进行验证。结果:对2000个突变体进行了筛选,共得到6株芽孢形成缺陷株,在LB培养基中培养5d后,镜下仍未见有芽孢形成,呈现明显的芽孢形成缺陷特征。Southern杂交表明野生株无杂交带,突变株均有且只有1条杂交带,且杂交带的位置不尽相同。结论:转座子Tn917可以单拷贝随机诱变炭疽杆菌野生株,产生在不同位点突变的突变株。  相似文献   

7.
微生物全基因组鸟枪法测序   总被引:4,自引:0,他引:4  
罗春清  杨焕明 《遗传》2002,24(3):310-314
全基因组测序主要有二种策略,一种是分级鸟枪法测序,另一种是全基因组鸟枪法测序。微生物是一种十分重要的遗传资源,运用全基因组鸟枪法可以方便、快捷地完成其基因组的测序任务。本文对微生物全基因组鸟枪法测序中文库构建、插入片段的长短比例、反应投入量、拼接以及补洞等问题作了较细致的描述,有些步骤作了举例说明。 Abstract:Two strategies introduced for whole genome sequencing,one is clone by clone method,the other is whole genome shotgun sequencing,for microbes which are very important to us,whole genome shotgun sequencing method is very convenient.In this article we discussed the library construction、long-to-short-ratio of insert,、total number of reads should be sequenced、assembly and gap filling technologies of the whole microbial genome shotgun sequencing method while some examples presented.  相似文献   

8.
A rapid and convenient method was proposed for constructing insertional mutants in the sequencing of extended DNA fragments. The gist is insertion of an antibiotic resistance gene in plasmid DNA digested with DNase I. DNase I provides for a uniform distribution of insertion sites along the plasmid, and the background is low owing to antibiotic-based selection. The method requires neither high quality nor large amounts of plasmid DNA (which is especially important with low-copied plasmids), yields the results independent of the plasmid nucleotide sequence, and allows highly accurate analysis and ordering of the insertional mutants.  相似文献   

9.
The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independent transformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.  相似文献   

10.
454测序技术在微生物生态学研究中的应用   总被引:1,自引:0,他引:1  
以Sanger法(双脱氧核苷酸末端终止法)为代表的第1代测序技术由于其成本高、速度慢、通量低等不足,满足不了大规模测序的要求.进入21世纪后,以Roche 454为代表的第2代测序技术诞生了,454测序法作为一种高通量的测序方法,近年来已被广泛应用于微生物生态学研究中.介绍了该测序技术的原理和操作步骤,结合本实验室的研...  相似文献   

11.
12.
微生物是人类赖以生存的重要资源,为提高微生物的生产效率或者赋予其新的生物学功能,需要通过理化方法进行诱变或通过分子生物学技术对其进行定点突变。在目前的理化诱变方法中,常压室温等离子(atmospheric and room temperature plasma,ARTP)诱变技术具有操作简单、条件温和、安全性高、诱变快速等优点,成为倍受青睐的新方法。基于此,综述了ARTP诱变技术的原理及其在微生物诱变育种方面的应用,以期为选育性能优越的微生物菌种的诱变育种相关研究提供借鉴。  相似文献   

13.
14.
在用散弹 (shotgun)法测定水稻 (OryzasativaL .ssp .indica)基因组全序列的过程中 ,叶绿体和线粒体DNA的污染问题非常严峻 .应用脉冲场电泳 (PFGE)技术对水稻基因组DNA进行纯化 ,结果表明它能够有效去除叶绿体和线粒体DNA ,使其污染率从 3%降低到 0 2 % .同时 ,比较了水稻绿苗和黄化苗的DNA得率 ,以及HB法和NIB法制备大分子质量(HMW)DNA的异同 .最后提出一套制备水稻基因组DNA的方法 ,包括黄化苗培养 ;细胞核的分离、包埋和裂解 ;脉冲场电泳纯化、回收聚集在低熔点 (LMP)胶中的水稻HMWDNA .用该方法所得的水稻基因组DNA ,纯度高 (无叶绿体和线粒体DNA污染 )、基因组完整 (机械剪切和降解少 )、回收率高 (操作过程中DNA损失少 ) .另外 ,首次报道在融化的低熔点(LMP)胶中对水稻HMWDNA于 38℃进行超声波处理 ,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1 5~ 3kb ,3~ 12kb) ,并且效果优于在TE中进行操作  相似文献   

15.
454测序法在环境微生物生态研究中的应用   总被引:3,自引:0,他引:3  
传统的Sanger测序技术虽已成熟,但其速度和成本的限制满足不了大规模测序的要求。第二代高通量测序技术结合了乳胶微粒和皮升级反应的454焦磷酸测序法,作为一种高通量测序技术,具有分析结果准确、高速、高灵敏度和高自动化的特点。对454测序法的技术原理和操作步骤进行了介绍,对近年来运用该方法在环境微生物生态研究领域的进展进行了综述。  相似文献   

16.
The identification of virulence factors in Rhodococcus equi has been severely hampered by the lack of a method for in vivo random insertion mutagenesis. This study reports the use of transposomes to generate random insertions of a gene conferring kanamycin resistance into the genome of R. equi ATCC 33701. Southern hybridisation using the kanamycin resistance gene as probe showed that insertion of transposome is random. This was confirmed following nucleotide sequence analysis of the junction between the transposome and chromosomal DNA. The presence of a 9 bp duplication of the target sequence showed that random integration of the transposome was due to a bona fide Tn5 transposition event.  相似文献   

17.
目的:研究和开发高通量测序全基因组组装过程中的填补gap的方法。方法:研究组装软件的算法,使用Perl语言编写自动填补gap的程序,并建立全基因组组装的流程。结果:提出了填补gap的末端延伸法,并使用Perl语言进行了编程;在对立克次体高通量测序的组装过程中,这些方法能大大减少gap的数量。结论:本研究提出的末端延伸法能够高效填补全序列组装过程中出现的gap,具有很强的实用性。  相似文献   

18.
Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis   总被引:8,自引:1,他引:8  
T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.  相似文献   

19.
The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The DNA sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a DNA fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.  相似文献   

20.
植物诱变技术是指利用外界因素加快物种遗传变异,在短期内获得有利用价值的突变体,为培育新种质、新品种及基因功能的研究等创造条件。相对于自然的选择,诱变技术具有高频率、广突变、周期短等特性。主要论述了常用的化学诱变、物理诱变、空间诱变和生物诱变等诱变手段,并详细介绍了上述诱变技术的诱变机理、生物学效应、常用的诱变方式及其在植物应用中的研究现状。针对现行各种诱变技术的不足提出了今后努力的方向。  相似文献   

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