利用FLP/frt重组系统产生无选择标记的转基因烟草植株

在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。     

高效删除标记基因的Bhlea2植物表达载体的构建

为培育去除选择标记基因的耐旱转基因植物,同时利用Cre/Lox和FLP/frt系统,构建一个能够高效删除标记基因的Bhlea2基因植物表达载体.拟南芥rd29A启动子是在低温、干旱、高盐胁迫下的快速应答启动子,玉米ubiquitin启动子可有效驱动外源基因的转录,拟南芥pAB5启动子是花粉及胚胎等发育早期特异表达的启动子,利用上述启动子构建了表达Bhlea2基因并能够删除标记基因的植物表达载体.该表达载体包括重组酶表达元件pAB5-FLP、Bhlea2抗旱基因表达元件rd29A-Bhlea2和bar标记基因表达元件ubiquitin-bar.     

ipt基因促进矮牵牛遗传转化效率及热激启动子驱动基因删除

本研究主要探讨ipt基因对矮牵牛遗传转化不定芽诱导影响及拟南芥热激启动子hsp18.2驱动重组酶基因flp的热诱导外源基因删除表达载体在矮牵牛中的基因删除效果.本研究中将植物表达载体pBin-hsp18.2:flp-35S:ipt及对照载体pBin-hsp18.2:flp遗传转化矮牵牛,以获得转基因植株,分析比较不定芽的诱导和转基因植株进行的热激基因删除.研究结果表明,ipt基因可促进矮牵牛遗传转化过程中不定芽的诱导,其不定芽诱导率为21.5%,显著高于对照的8.7%.在44℃,6 h,热激6次的条件下,转基因矮牵牛植株表型恢复正常,经 GUS蛋白表达分析及PCR、RT-PCR检测,证明外源基因已经被删除.转基因矮牵牛基因删除效率最高可达43.8%.本研究为ipt基因在一些遗传转化困难植物转基因中的应用奠定了基础.     

利用Cre/lox重组系统获得无选择标记的SKTI抗虫转基因烟草

将置于两个同向lox位点之间的Bar基因表达盒与大豆胰蛋白酶抑制剂SKTI基因表达盒融合后获得相应植物表达载体,转化烟草Wisconsin 38后获得对棉铃虫具有明显抗性的SKTI转基因植株。SKTI转基因植株通过叶盘二次转化法导入Cre基因,对再生植株叶盘进行Basta的抗性检测,检测Bar基因的删除情况。结果表明:绝大多数再生植株对应叶盘在含8 mg/L PPT的筛选培养基上无法再生,Bar基因被删除的效率在38%~100%之间。对Bar基因删除区域进行PCR及克隆测序后发现Bar基因表达盒被精确删除。对Bar基因删除植株开花自交获得的分离后代进行NPTⅡ抗性检测,5株NPTⅡ敏感植株分子检测显示均只含有SKTI基因而无Cre基因存在,为无选择标记基因的SKTI转基因植株。     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17.5C控制Cre定位重组酶介导的DNA删除系统.在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S-GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除.通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率.结果显示在两个小时热激处理后转基因烟草中有41%的CaMV35S-GUS片段被删除.由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作.     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17．5C控制Cre定位重组酶介导的DNA删除系统。在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S—GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除。通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率。结果显示在两个小时热激处理后转基因烟草中有41％的CaMV35S—Gus片段被删除。由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作。     

培育无选择标记的转基因植物

目前,几乎所有的植物遗传转化都要通过使用选择标记基因,如抗生素或除草剂抗性基因等来筛选转化子,虽然没有研究结果表明选择标记基因影响人类健康或环境安全,但近年来也引发了人们对转基因产品安全性的担心。为了消除公众对转基因食品的安全性顾虑,无选择标记的转基因植物应运而生。本文综述了共转化系统、位点特异性重组系统(包括FLP/FRT、Cre/lox、R/RS及Gin/gix系统)和转座子系统(Ac/Ds转座子系统)在培育无选择标记转基因植物中的应用。     

含有融合标记基因和LoxP/FRT位点的植物表达载体构建及应用

本研究利用化学合成法合成了包含pCAMBIA0390左右边界T-DNA和LoxP/FRT (LF)位点的DNA片段Ⅰ,利用SacⅡ和SphⅠ酶切位点,去除了pCAMBIA0390左右边界T-DNA和多克隆位点之间的序列,然后连接DNA片段Ⅰ和载体片段,构建了植物表达载体pGM323-LF-enTP.随后,再合成含有适合在单子叶植物中表达的由玉米Ubi-1启动子驱动的融合标记基因(Bar::gus)和水稻actin-1启动子驱动的Bt抗虫蛋白基因(Cry1Ab)表达元件的DNA片段Ⅳ,在pGM323-LF-enTP的基础上,利用SalⅠ和PstⅠ位点构建了同时含有LF位点、Bar::gus以及Cry1Ab基因表达元件的表达载体pGM626-LF-ABt.利用含有pGM626-LF-ABt的农杆菌遗传转化烟草和玉米,以草丁膦作为抗性筛选剂,非转化细胞得到了有效抑制,快速获得了转基因植株,利用GUS组织化学检测和RT-PCR分析了转基因植株中标记基因的表达,结果表明pGM626-LF-ABt可以用于农杆菌介导的单、双子叶植物遗传转化.本研究为培育安全抗虫转基因植物奠定了基础.     

Candida amazonensis FLP/FRT基因敲除系统的构建及初步验证

【目的】构建一个适用于Candida amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并通过敲除C.amazonensis的丙酮酸脱羧酶基因(Pyruvate decarboxylase,PDC)对该系统进行初步验证。【方法】以gfpm(绿色荧光蛋白基因)为报告基因,通过添加相应诱导剂评估Spathaspora passalidarum来源启动子(SpXYLp、SpMAL6p、SpMAL1p、SpGAL1p)和Saccharomyces cerevisiae来源Sc GAL1p启动子在C.amazonensis中的诱导调控性能。选择严格诱导型启动子调控FLP重组酶的表达,并在FLP表达盒和潮霉素(Hygromycin B)抗性标记基因(hphm)两端添加同向重复的FRT位点,以PDC基因作为靶基因构建敲除盒PRFg HRP,转化宿主菌C.amazonensis CBS 12363,筛选得到阳性转化子后,通过添加诱导剂,表达FLP重组酶,实现FRT位点间片段切除。【结果】诱导调控实验表明启动子SpGAL1p(受半乳糖诱导)和SpMAL1p(受麦芽糖诱导)是适用于C.amazonensis的严格诱导型启动子。以SpGAL1p调控FLP基因表达,构建的敲除盒PRFg HRP成功转化宿主菌,获得阳性转化子C.amazonensis PDC01,通过添加半乳糖诱导,成功切除基因组中FLP表达盒和抗性标记盒,获得突变株C.amazonensis PDC02。【结论】首次建立了一个适用于C.amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并利用该系统成功敲除了C.amazonensis内的PDC基因,为进一步利用代谢工程改造C.amazonensis酵母奠定了良好基础。     

木糖异构酶基因xylA的克隆及其表达载体的构建

木糖异构酶基因xylA是一种正向选择标记基因,在植物基因工程中使用该标记可以获得安全的转基因植物.构建了以xylA基因为选择标记的植物表达载体.从大肠杆菌Top10中扩增出xylA基因,插入到质粒pCAMBIA2301的Xho Ⅰ位点,通过酶切和PCR检测插入片段的正确性,得到载体pCAMBIA2301-xylA,将pBI121载体上的‘35S-GUS-Nos'表达框插入到pCAMBIA2301-xylA的EcoR Ⅰ和Hind Ⅲ位点.得到中间载体pCAMBIA2301-xylA-GUS,用Sac Ⅰ和Sma Ⅰ切下克隆载体上的CBF1基因替代pCAMBIA2301-xylA-GUS中的GUS片段,用电转化法将获得的表达载体转化到农杆菌中,为将来获得安全的转基因抗寒植株奠定基础.     

Production of Marker-free Transgenic Tobacco Plants by FLP/frt Recombination System

Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of 2-μm plasmid from Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt-containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat-shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41 % re-transgenic tobacco plants, which indicated that this system could make a great contribution obtaining the marker-free transgenic plants.     

Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter

To excise a selectable marker gene from transgenic plants, a new binary expression vector based on the 'genetically modified (GM)-gene-deletor' system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Histochemical staining for GUS activity showed that, upon induction by heat shock, all exogenous DNA, including the selectable marker gene ipt, beta-glucuronidase (gusA) gene and the FLP recombinase gene, between two loxP/FRT sites was eliminated efficiently from primary transgenic tobacco plants. Molecular analysis further confirmed that excision of the marker gene (ipt) was heritable and stable. Our approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants.     

Cre/lox位点特异重组系统在转基因植物中的应用

位点特异重组系统由重组酶和相应的重组酶识别位点组成,通过两者间的相互作用,实现外源基因精确整合与切除等一系列遗传操作.主要可分为Cre/lox系统、FLP/frt系统、R/RS系统和Gin/gix系统.目前,研究最充分应用最广泛的位点特异重组系统为Cre/lox系统.此系统为位点特异重组系统家族中的一员,由38.5kDCre重组酶和34bplox位点组成,最早被应用于动物转基因研究,包括基因敲除、基因激活、基因易位等.近年来,随着研究的深入,Cre/lox系统被逐步应用到植物研究中,并在诸多领域取得重大进展.本文总结归纳了Cre/lox系统在定点整合、定点切除以及叶绿体转化等方面的最新研究成果,旨在为利用Cre/lox系统构建环境安全和高效表达的植物遗传转化体系提供参考.     

A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants

SUMMARY: The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-microm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Utilization of PVX-Cre expression vector in potato

Trait genes are usually introduced into the plant genome together with a marker gene. The last one becomes unnecessary after transgene selection and characterization. One of the strategies to produce transgenic plants free from the selectable marker is based on site-specific recombination. The present study employed the transient Cre-lox system to remove the nptII marker gene from potato. Transient marker gene excision involves introduction of Cre protein in lox-target plants by PVX virus vector followed by plant regeneration. Using optimized experimental conditions, such as particle bombardment infection method and application of P19 silencing suppressor protein, 20-27% of regenerated plants were identified by PCR analysis as marker-free. Based on our comparison of the recombination frequencies observed in this study to the efficiency of other methods to avoid or eliminate marker genes in potato, we suggest that PVX-Cre mediated site-specific excisional recombination is a useful tool to generate potato plants without superfluous transgenic sequences.     

美洲商陆抗真菌蛋白转化烟草的研究和抗病性检测

本研究为美洲商陆抗病毒蛋白(PaAFP)基因首次对植物遗传转化的研究,转入烟草中研究此蛋白对烟草立枯病的抗性。从美洲商陆叶片中获得美洲商陆抗真菌蛋白前体蛋白基因cDNA序列,构建植物表达载体pCAMBIA1300-PaAFP,通过三亲杂交法将其导入根癌农杆菌LBA4404受体菌,转染烟草获得了大量再生转基因植株。PCR、Southern杂交、RT-PCR以及Tris-Tricine-SDS-PAGE检测结果表明目的基因已经整合到烟草基因组中,并且已经得到转译。转基因植株苗期抗立枯病试验表明,转基因烟草植株对立枯丝核菌表现出了抗性。     

FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.