利用Tn5转座子构建杆状病毒AcMNPV随机突变体的初步研究

以杆状病毒模式种AcMNPV为研究对象,应用基于Tn5转座子的随机转座的方法,构建杆状病毒突变体库将果蝇hsp70启动子后接绿色荧光蛋白基因后插入Tn5转座子,构建了可以在昆虫细胞中表达,易于跟踪的转座载体．利用体外转座系统将转座子随机插入AcMNPV基因组,并用转座反应液转染Sf21细胞,得到了表达绿色荧光蛋白的病毒突变体库进一步纯化了两株病毒B9F和Li6A,进行了转座子插入位点的分析,确定两株病毒中,转座子分别插入了94K基因和p10基因．该方法将为杆状病毒功能基因组研究提供重要的手段。     

piggyBac共转染系统的构建以及转座活性的验证

piggyBac(PB)转座子的末端反向重复序列(ITR)和转座酶编码序列(PBase)分别克隆于质粒PB1156、atub-pBac-K10. ITR与包含红色荧光蛋白表达序列(RFP)的质粒pmCherry-N1重组构成供体质粒(Donor);PBase与真核表达载体pEFrF-PGK重组构成辅助质粒(Helper).使用由供体质粒与辅助质粒组成的PB二元共转染系统转染HEK293细胞,结果表明该二元共转染系统具有较好的转座活性,并且在分子水平验证了转座的发生以及使用反向PCR(IPCR)定位了其中一     

铜绿假单胞菌鞭毛运动相关基因的研究

建立优化的转化条件,将M u转座复合物电转化到临床分离的一株铜绿假单胞菌(P seud om onasaerug inosa)PA 68中,最高转化效率达3.66×104CFU/μg DNA.通过表型筛选,得到三株鞭毛运动能力缺陷的突变子,Sourn thern杂交证实转座子为单点插入.经基因克隆、核苷酸测序研究,证明转座子分别插入到uvrD、phzF 1、zw f三个基因中,这是首次在国际上将M u转座重组技术应用到鞭毛运动相关基因的研究中.由于人工M u转座技术具有随机单点插入的优点,克服了传统转座子能在染色体上迁移的缺点,为进一步研究P.aerug inosa的鞭毛运动机理及致病性奠定基础.     

利用加强型绿色荧光蛋白标记黄色单胞菌的研究

通过连接T载体与来自质粒pSEBG上的egfp基因而构建成质粒pZTE,并将质粒pZTE的egfp基因插入到自杀质粒pTnMod-OGm转座子上的多克隆位点Kpn I和Sal I中构建成质粒pTE-OGm,最后pTE-OGm的egfp基因转座子元件通过转座作用插入到受体菌黄色单胞菌L1的染色体中,从而使黄色单胞菌L1获得较稳定的绿色荧光标记,为进一步在MBR反应器中更好地示踪黄色单胞菌L1的生长和迁移情况奠定了基础。     

建立转座子用于原代树突状细胞转染体系的研究

应用PCR技术从p EGFP-N1载体扩增获取CMV启动子和EGFP基因,利用限制性内切酶BglⅡ/NotⅠ以及NotⅠ/Eco RⅠ将其插入p T/HB,构成p T-CMV-EGFP重组转座子,p T-CMV-EGFP与SB11转座酶形成"睡美人"转座系统.采用脂质体与荧光表达载体p EGFP-N1不同配比转染小鼠成纤维细胞系3T3,p TCMV-EGFP与SB11梯度比例转染小鼠肝癌细胞系H22后,经流式细胞术比较阳性细胞率,当条件为脂质体与总DNA质量比为4∶1;转座酶与转座子间质量比为1∶4转染时,细胞阳性率最高(H22悬浮细胞可达42.23%),转染结果较稳定(SD=1.25).通过此优化体系转染小鼠骨髓来源的树突状细胞DCs,经激光共聚焦显微镜显示,能够获得被EGFP修饰的DCs,并通过流式细胞术检测被标记有EGFP的特异DCs阳性率可达20%以上.表明本研究已成功建立了一个可用于原代DCs转染的方法.     

Tn7转座子在大肠杆菌、迟钝爱德华氏菌及鳗弧菌中的应用

Tn7转座子是一种定点插入型转座子,固定地插在细菌的glmS基因下游的attTn7位点,而glmS基因与细胞壁的合成有关,存在于所有的细菌中,所以Tn7转座子可以插入到所有细菌的染色体,因此Tn7转座系统已被开发为细菌基因组改造的重要工具。为了方便对大肠杆菌、迟钝爱德华氏菌和鳗弧菌的改造和研究,探讨应用Tn7转座子对其基因组进行改造的操作步骤,并利用阿拉伯糖启动子pBAD诱导的Flp/FRT系统建立了高效、快捷的抗性消除方法。     

Tn5转座子插入p95基因不影响AcMNPV的复制

从Tn5转座子介导的AcMNPV随机插入突变体库中,分离到一株复制正常的突变体AcApra41.突变定位发现Tn5转座子插入了病毒p95基因中.为了排除AcApra41中还有其他突变,利用同源重组法构建了p95基因定点插入突变的重组病毒AcGFP-P95in.PCR确认p95基因中插入了Tn5转座子;Western blot也证实AcApra41和AcGFP-P95in感染的细胞中,P95蛋白的分子量都因为插入突变而变小,由野生型的95 ku变为 55 ku.病毒复制动态曲线和荧光显微镜观察证实带有该插入突变的病毒能够在Sf9细胞中正常复制,并表达极晚期基因.这一结果表明完整的P95蛋白对病毒复制是非必须的.     

在中国做一流的研究——访复旦大学发育生物学研究所许田、吴晓晖

去年8月,世界一流的生命科学杂志——美国《细胞》杂志(《Cell》杂志)的封面吸引了海内外众多专家、学者以及科学爱好者们的注意:几只转基因小鼠在漆黑的实验室里闪闪发光,红色泛蓝的荧光毛皮使它和制造它的中国科研人员一起登上了国际顶级生命科学杂志的封面。复旦大学发育生物学研究所的科研人员将一种源于飞蛾的PB(PiggyBac)转座子(Transposon)用于小鼠和人类细胞的基因功能研究,在世界上首创了一个高效实用的哺乳动物转座因子系统,为大规模研究哺乳动物基因功能提供了崭新途径。相关技术已申请国际专利。他们的科研成果入选“2005中国高校科技十大进展”,和这一成果同样引人注目的是其国际领先的研究方法和一流的科研团队。     

Spexin转基因小鼠的构建及表型初步分析

Spexin是一种具有多种生物学功能的神经肽,在代谢疾病及神经系统疾病中发挥重要作用.为了深入研究spexin的生理功能和相关机制,构建了spexin转基因小鼠,通过检测血液中spexin含量以及糖耐受实验,对模型小鼠进行了初步的表型分析.利用piggyBac(PB)转座子技术培育spexin转基因小鼠,采用聚合酶链反应(polymerase chain reaction,PCR)和酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)分别在基因层面和蛋白层面验证了模型动物是否构建成功.之后用饲喂高脂饲料的雄性小鼠进行糖耐受实验,对转基因模型小鼠进行表型分析.PCR结果显示,以靶向spexin的两对引物扩增,转基因型小鼠基因组DNA中能分别扩增出300 bp和301 bp的片段,而野生型小鼠则不能.ELISA结果中,转基因型小鼠血清中spexin含量较野生型小鼠显著升高;同一表型中,雌雄小鼠间血清中spexin水平无明显差异.饲喂高脂饲料85 d内,转基因型小鼠和野生型小鼠体重变化未观察到显著差异;糖耐受结果显示,转基因型小鼠每个时间点血糖浓度均低于野生型小鼠,且在15 min时具有显著性差异.结果显示成功构建了过表达spexin基因的小鼠模型.spexin表达水平的升高在一定程度上提高了C57BL/6小鼠的糖耐受能力.该模型为进一步研究spexin基因在动物体内的功能以及在代谢疾病发病机制中的作用奠定了基础.     

植物分子生物学中的双元系统

现代植物分子生物学理论研究和实际应用,发展了许多双因子系统即双元系统,其中具有代表性的有双元载体系统和双元标签系统.双元载体系统的构建是基于T-DNA的转移机制,由两种质粒组成,一种为穿梭载体,另一种为辅助载体.双元标签系统是以Ac转座子和Ds转座子构建的.Ac/Ds转座系统在高等植物基因的识别、克隆和对植物发育过程的基因表达中广泛应用.     

Transpositional behaviour of the Ds element in the Ac/Ds system in rice

Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a ma- jor approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA trans- formed homozygotes were crossed with Ac-T-DNA transformed homozygotes in order to establish an Ac/Ds transposon system in rice. The successive investigation of Ds transposition from F1 to F5 gen- erations indicated that the frequencies of germinal transposition increased over successive genera- tions and reached 54.2% in F3 generation. The Ds transposition pattern revealed that a Ds transposition induced an approximately 170-bp deletion of T-DNA sequence and another Ds transposition carried a 272-bp T-DNA sequence. Using thermal asymmetric interlaced PCR (TAIL-PCR), some flanking se- quences of the Ds element were amplified. Analyses of 17 Ds-flanking sequences showed that five Ds were inserted into gene regions. The Ds could transpose not only to the linked sites but also to the unlinked sites. The frequency of inter-chromosomal transposition of Ds was 33.3%.     

拟南芥雄性不育基因ds254的定位及其表达模式

通过Ac／Ds转座子系统的诱变，得到拟南芥突变体DS254．它的发育进程比野生型慢，植株比野生型矮小，连座叶柄比野生型的短，花苞数目比野生型的多，分枝的数量比野生型的多，且雄性不育．遗传分析表明突变体属于隐性单基因突变，稳定遗传，并且与Ds是紧密连锁的。可能是Ds插入直接引起的突变．TAIL—PCR的方法将该基因定位在第4条染色体上．Ds上携带的GUS基因表迭的初步分析表明，GUS基因在突变体的花药和连座叶中表达，说明该基因也可能在这些部位表达．突变体的表型分析结果说明该基因可能在植物的许多发育过程中起作用．     

Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line.

Transposons have been enormously useful for genetic analysis in both Drosophila and bacteria. Mutagenic insertions constitute molecular tags that are used to rapidly clone the mutated gene. Such techniques would be especially advantageous in the nematode Caenorhabditis elegans, as the entire sequence of the genome has been determined. Several different types of endogenous transposons are present in C. elegans, and these can be mobilized in mutator strains (reviewed in ref. 1). Unfortunately, use of these native transposons for regulated transposition in C. elegans is limited. First, all strains contain multiple copies of these transposons and thus new insertions do not provide unique tags. Second, mutator strains tend to activate the transposition of several classes of transposons, so that the type of transposon associated with a particular mutation is not known. Here we demonstrate that the Drosophila mariner element Mos1 can be mobilized in C. elegans. First, efficient mobilization of Mos1 is possible in somatic cells. Second, heritable insertions of the transposon can be generated in the germ line. Third, genes that have been mutated by insertion can be rapidly identified using inverse polymerase chain reaction. Fourth, these insertions can subsequently be remobilized to generate deletion and frameshift mutations by imperfect excision.     

Transposition of Tn554 does not generate a target duplication

Transposable elements from prokaryotic and eukaryotic organisms are discrete DNA segments bounded by inverted or directly repeated sequences that insert into non-homologous DNA in a reaction that is independent of the general recombination functions of the host. The mechanisms proposed generally involve a staggered double-stranded scission of the target DNA, ligation to the nicked ends of the transposable element, and replication of the element, resulting in the generation of a directly repeated oligonucleotide target sequence flanking the new copy of the element. Most transposons have a relatively low degree of target site specificity coupled with a low insertion frequency. Tn554, a Staphylococcus aureus transposon which specifies resistances to erythromycin and spectinomycin, displays an unusually high degree of insertion specificity. Tn554 transposes with high efficiency to a unique ('primary') site in the S. aureus chromosome and only rarely (less than 10(-6) per transductant) to other, secondary sites. We report here the nucleotide sequences surrounding the junctions of Tn554 in three independent 'primary' insertions and two 'secondary' insertions of the transposon. Two unusual features are revealed: first, the termini of Tn554 contain neither inverted nor directly repeated sequences. Second, transposition of Tn554 does not generate the short direct repeats of the target DNA that are characteristic of other transposable elements. These results suggest that the mechanism of Tn554 insertion may be significantly different from that of other transposons.     

DNA sequence at the end of IS1 required for transposition

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.     

Construction and genetic analysis of mutator insertion mutant population in maize

A total of 26718 M1 plants were ob- tained by crossing the active mutator transposon donor parents (Q105, WW51, 115F, V26-2 and 919J) with the recipient parents (Hz85,W328 with Bz gene and S-Mo17Rf3Rf3). The phenotypes of M1 plants were observed in the field. M1 plants were self-pollinated to develop the mutator insertion-mutagenized M2 seeds. The transposition frequency of the mutator in the genome was calculated based on the spotted aleurone phenotype of the M2 seeds. The results showed that: (1) the mutation frequency of M1 phe- notypes in the field was 0.07 in the population of W328×Mu; (2) the mutation frequency of spotted aleurone seeds on the M2 ears was 0.122 in the population of W328×Mu; (3) five S-cytoplasm male-sterile plants were found among 22500 M1 plants of S-Mo17Rf3Rf3×Mu, with the transposition frequency about 2.2×10?4 per locus. 99 flanking se- quences of mutator transposition were amplified by the modified MuTAIL-PCR, and 59 non-redundant sequences with length around 400 bp were obtained. After bioinformatic analysis, 27 sequences of them could be annotated, using non-redundant nucleotide database of maize, rice, and Arabidopsis. 36 se- quences of them were located on the genetic map of maize by comparative genomics, and several flank- ing sequences of mutator insertion were mapped on the single marker locus. Hotspot sequences of mu- tator transposition were revealed by comparing the homologies between the 9-bp target site duplication of the mutator insertion. The putative functions of 8 flanking sequences of mutator transposition had identity with the functions of their correspondingmarker. The constructed mutator insertion mutant population in maize will facilitate the new gene discovery and functional genomics study in maize.