Development of disease-resistant marker-free tomato by R/RS site-specific recombination

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).     

Generation of selectable marker-free transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system

The aim of this research was to generate selectable marker-free transgenic tomato plants with improved tolerance to abiotic stress. An estradiol-induced site-specific DNA excision of a selectable marker gene using the Cre/loxP DNA recombination system was employed to develop transgenic tomato constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase gene from Arabidopsis thaliana. Transgenic tomato plants containing a selectable marker were also produced as controls. The expression of AtIpk2β conferred improved resistance to drought, cold and oxidative stress in both sets of transgenic tomato plants. These results demonstrate the feasibility of using this Cre/loxP-based marker elimination strategy to generate marker-free transgenic crops with improved stress tolerance.     

Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene

Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.     

Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system

Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a tobacco cell. When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre-loxP site-specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid-targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation (Transformation Protocol), by pollination (Pollination Protocol) or from a transient, non-integrated T-DNA (Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10-16 months, depending on which protocol is used.     

A system to induce the deletion of gemomic sequences using R/RS site-specific recombination and the Ac transposon in transgenic rice plants

Recombinase encoded by the R gene of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce deletion or inversion of the DNA segment that is flanked by the RSs. The R gene under the control of the CaMV 35 S promoter was introduced into rice (Oryza sativa L.). R/RS-specific deletion was first demonstrated in transgenic rice callus carrying the R gene by transient introduction of a cryptic reporter gene that was designed to confer β-glucuronidase (GUS) expression once deletion between two RSs took place. The rice containing the R gene was subsequently crossed with transgenic rice carrying (I-RS/dAc-I-RS) T-DNA that contained RS sequences within the T-DNA and another RS in a modified Ac element that had been transposed to a new locus by Ac transposase. Deletion of the gemomic sequences flanked by the two RSs was detected by PCR analysis in somatic cells of F₂ plants. These results demonstrate a technical advance in that the R/RS recombination system, in combination with the Ac transposable element, can be used to generate deletion in rice chromosomes. Received: 30 June 2000 / Accepted: 16 October 2000     

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.     

Auto-excision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT site-specific recombination system

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13–41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.     

Use of site-specific recombination to regenerate selectable markers

Summary A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated. This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 m circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs. When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats. In the example described, the S. cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P. pastoris strain.     

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.     

In vivo site-specific recombination using the beta-rec/six system

The prokaryotic beta serine recombinase (beta-rec) catalyzes site-specific recombination between two directly oriented six sites (93 bp) in mammalian cells, both in episomal and in chromosomally integrated substrates. The beta-rec/six exclusive intramolecular site-specific recombination (SSR) system has been proposed as a suitable approach when several independently controlled recombination events are needed in a single cell. Here we explored the use of the beta-rec/six system for selective induction of genome-targeted modifications. We generated and analyzed mouse transgenic lines (Tgbeta) expressing beta-rec under the control of the Lck promoter. beta-rec activity was demonstrated, and there was no evidence of alterations to thymic or peripheral T cell development. We developed two transgenic mouse lines harboring different target sequences (Tgrec and KOsix) and analyzed the effect of beta-rec expression on these animals. The results indicate that the beta-rec/six SSR system is functional for in vivo gene-targeting applications.     

利用子房滴注法获得无载体骨架序列和选择标记的转基因玉米

转基因植物中的载体骨架序列和选择标记基因是引起生物安全性争论的根本原因, 最直接、最有效的解决方法是在转化过程中不使用载体骨架序列和选择标记基因。本研究建立并优化了玉米子房滴注转化法, 其操作要点是将DNA转化溶液直接滴加在完全去除花柱的子房上。利用子房滴注法将无载体骨架序列和选择标记的线性GFP基因表达框转化玉米。PCR结果表明: 适合子房滴注法转化的玉米品种为9818, 最佳转化时间为授粉后18~20 h, 在此条件下得到最高的PCR阳性率, 为3.01%; Southern blotting结果表明外源基因的整合方式简单(1~2条杂交带); RT-PCR结果表明转基因植株中GFP基因能够在RNA水平上正常表达; 在转基因植株的根和幼胚中观察到GFP表达。     

Development of selectable marker free,insect resistant,transgenic mustard (Brassica juncea) plants using Cre/loxmediated recombination

β-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries. We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742∆suc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme. The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.     

Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.     

Production of marker-free transgenic Nierembergia caerulea using MAT vector system

Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt ⁻, GUS⁺ and excision⁺ as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents.     

Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker

A plant transformation vector, pCLKSCLA25 (EU327498), was developed to contain eight cloning sites and the inducible self-excision system which provided an effective approach to eliminate the selectable marker gene(s) from transgenic plants. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding the selectable marker neomycin phosphotransferase and cre itself. The excision efficiency was 41% in transgenic tomato regenarants. The stilbene synthase gene (vst1) from Vitis vinifera L. was cloned into pCLKSCLA25. The expression of vst1 gene contributed to the accumulation of trans-reveratrol from 3.4 to 8.7 μg/g fresh wt in different marker-free transgenic tomato lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.     

利用CRISPR/Cas9技术构建定点突变小鼠品系

CRISPR/Cas9技术是新近发展起来的对细胞和动物模型进行基因编辑的重要方法。本文利用DNA双链断裂(Double-strand breaks, DSBs)引起的同源重组(Homologous recombination, HR)依赖与非依赖的修复机制,建立基于CRISPR/Cas9核酸酶技术构建定点突变小鼠品系的技术体系。针对赖氨酸特异脱甲基化酶2b(Lysine (K)-specific demethylase 2b, Kdm2b)酶活关键位点对应的基因组DNA序列设计单一导向RNA(Single-guide RNA, sgRNA),通过与Cas9 mRNA共显微注射,分别得到Kdm2b基因发生移码突变的基因失活品系及关键位点氨基酸缺失的酶活突变型小鼠品系。此外,利用HR介导的修复机理,将黄素单加氧酶3(Flavin containing monooxygenases3, Fmo3)基因的sgRNA序列及对应的点突变单链寡脱氧核苷(Single strand oligonucleotides, ssODN)修复模板共注射到小鼠受精卵雄原核。对F₀小鼠基因测序分析显示,成功构建了Fmo3基因移码突变的基因敲除和单碱基定点突变的基因敲入小鼠,这些突变能够稳定遗传给子代。本研究利用CRISPR/Cas9技术,通过同源重组依赖与非依赖两种DNA损伤修复方式,成功构建了特定位点突变的小鼠品系。     

Production and selection of marker-free transgenic plants of <Emphasis Type="Italic">Petunia hybrida</Emphasis> using site-specific recombination

MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately 4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1–3 for ipt gene and 1–2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium.