Specific features of vector sequences insertion in the genome of transgenic plants

Upon processing of T-DNA, both borders may be recognized incorrectly. A number of transgenic plants containing, apart from a fragment of vector DNA adjacent to the left border of T-DNA, a vector fragment inserted irrespective of it were identified. Sequence analysis confirmed that genes located beyond the boundaries of T-DNA are transferred and inserted in the genome of transgenic plants. Recombination of vector DNA was discovered among plants of the first generation after self-pollination of the original transformants.     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。     

Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration

During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nos-nptll gene within the T-DNA and a mas2'-gusA (β-glucuronidase) gene outside the T-DNA borders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gusA gene and for the presence of binary vector backbone sequences. Approximately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.     

Genetic analysis of T-DNA insertions into the tobacco genome

A genetic test was performed on seeds from 283 transgenic tobacco plants obtained by T-DNA transformation. Seeds from self-fertilized transgenic plants were germinated on kanamycin-containing medium, and the percentage of seeds which germinated, as well as the ratio of kanamycin-resistant to kanamycin-sensitive seedlings were scored. Nine categories of transformants could be distinguished according to the number of loci into which T-DNA had inserted, and according to the effects of T-DNA integration on seed or seedling development. In most of the plants, T-DNA was inserted into a single site; others contained multiple independent copies of T-DNA. The number of T-DNA integration sites was found to be independent of whether a binary vector system or a cointegrate Ti plasmid had been used to obtain the transgenic plant. Loss of marker genes or marker gene expression from generation to generation appeared to be a quite frequent event. Plants which appeared to be insertional recessive embryo-lethal mutants did not exhibit this trait in the next generation.Abbreviations Kan^R kanamycin resistant - Kan^S kanamycin sensitive - NOP nopaline - NOS nopaline synthase - NPT II neomycin phosphotransferase II     

Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA.

For the screening of transfer DNA (T-DNA) integration in transgenic plant material, we developed a method based on specific amplification of genomic plant DNA flanking T-DNA borders. This approach is possible because the length of the region flanking T-DNA extremity on a restriction fragment is specific to the integration locus. We have modified an adaptor ligation PCR technique developed for amplification of unknown DNA flanking known sequence. The PCR patterns obtained were specific and reproducible for different plants from a given transgenic line. Furthermore, the number of PCR products obtained could be considered a good estimation of the T-DNA copy number. When compared to Southern blot analysis, the PCR results give valuable complementary information about the complexity of the T-DNA integration pattern and also about the integrity of the T-DNA borders. We describe the applications of this approach to populations of transgenic Arabidopsis thaliana plants.     

水稻T-DNA插入突变体库的构建及突变类型的分析

利用农杆菌介导的转化系统转化中花11成熟胚愈伤组织,获得1489个独立转化的T-DNA插入再生株系。PCR和Southern杂交的结果表明,69．8％转化株系被整合了T-DNA,通过Tail-PCR也从转化植株中扩增出T-DNA侧翼序列。同时对1066个T1转化株系的抽穗期、株高、单株穗数的调查结果表明,不同株系中分离出了突变植株。     

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.     

Molecular characterization of T-DNA integration sites in transgenic birch

The integration and structure of a transgene locus can have profound effects on the level and stability of transgene expression. We screened 28 transgenic birch (Betula platyphylla Suk.) lines transformed with an insect-resistance gene (bgt) using Agrobacterium tumefaciens. Among the transgenic plants, the copy number of transgene varied from one to four. A rearrangement or partial deletion had occurred in the process of T-DNA integration. T-DNA repeat formation, detected by reverse primer PCR, was found among randomly screened transgenic lines. Sequencing of the junctions between the T-DNA inserts revealed deletions of 19–589 bp and an additional 45 bp filler DNA sequence was inserted between the T-DNA repeats at one junction. Micro-homologous sequences (1–6 bp) were observed in the junctions between the T-DNA inserts. Using SiteFinding-PCR, a relatively high percentage of AT value was found for the flanking regions. Deletion of the right border repeat was observed in 12/18 of the T-DNA/plant junctions analyzed. The number of nucleotides deleted varied from 3 to 712. Deletions of 17–89 bp were observed in all left T-DNA/plant junctions analyzed. A vector backbone DNA sequence in the transgene loci was also detected using primer pairs outside the left and right T-DNA borders. Approximately 89.3% of the lines contained some vector backbone DNA. These observations revealed that it is important to check the specificity of the integration. A mechanism of T-DNA transport and integration is proposed for this long-lived tree species.     

Construction of Plant Expression Vector with Double Resistance to Virus and Insect and Identification of Transformation in Tomato

By in situ hybridization of bacterium clone and analysis of restriction enzyme digestion, both CMV-cp gene and Bt-toxin gene were inserted one by one into T-DNA of binary plant expression vector pea. The reconstructed plasmid was named pE14. Then, tomato was transformed with pE14 mediated by Agrobacterium tumefaciens GV311-SE, four regenerated tomato plants were obtained on the MS medium containing 100 μg/mL kanamycin. Assay of nopaline, dot blotting of tomato genomic DNA and PCR amplication of CMV-cp gene and Bt-toxin gene from genomic DNA showed that CMV-cp gene and Bt-toxin gene were transferred into the four regenerated tomato plants simultaneously with T-DNA, and no recombination of genes occurred. RNA dot blotting showed that two of them could express simultaneously the CMV-cp gene and Bt-toxin gene proteins. The resistances to virus and insect of the transgenic tomato plants will be tested in their F1 and F2 regenerations.     

T-DNA insertional mutagenesis for functional genomics in rice

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.     

T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation

Transgenic Arabidopsis and tobacco plants (125) derived from seven Agrobacterium-mediated transformation experiments were screened by polymerase chain reaction and DNA gel blot analysis for the presence of vector `backbone' sequences. The percentage of plants with vector DNA not belonging to the T-DNA varied between 20% and 50%. Neither the plant species, the explant type used for transformation, the replicon type nor the selection seem to have a major influence on the frequency of vector transfer. Only the border repeat sequence context could have an effect because T-DNA vector junctions were found in more than 50% of the plants of three different transformation series in which T-DNAs with octopine borders without inner border regions were used. Strikingly, many transgenic plants contain vector backbone sequences linked to the left T-DNA border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. We assume that integration into the plant genome of complete vector backbone sequences could be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model would imply that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer.     

Gene targeting in plants using theAgrobacterium vector system

In the past decade several methods have been developed for the introduction of foreign DNA into plant cells to obtain transgenic plants. In some of these methods, purified DNA is directly introduced into protoplasts that for some species can be regenerated into mature plants. The more commonly used protocols, however, employ the natural capacity ofAgrobacterium tumefaciens to transfer a defined peice of DNa, called T-DNA, to the nucleus of plant cells that are more easy to regenerate than protoplasts. In plant cells, like in animal cells, foreign DNA (including T-DNA) is readily inserted into the genome via illegitimates recombination. In contrast, targeted integration via homologous recombination, referred to as ‘gene targeting’, can only be obtained at relatively low frequencies. Nevertheless, gene targeting has become a standard strategy for reverse genetics studies in animals. In plants, the occurrence of gene targeting was only reported recently. This review focuses on the use of theAgrobacterium vector system to achieve gene targeting in plants. Recent experimental data concerning gene targeting in plants are presented and the overall suitability ofAgrobacterium T-DNA transfer for this purpose is assessed in light of contemporary views on the mechanism of T-DNA transfer.     

Development of a highly effective T-DNA inserted mutant screening method in a Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. spp. <Emphasis Type="Italic">pekinensis</Emphasis>) reverse genetics system

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1–1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.     

Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.)

A system for the genetic transformation of rye by co-cultivation with Agrobacterium tumefaciens is described. A total of 45 independent transgenic plants were regenerated with a transformation efficiency of 1 to % of the inoculated explants. The co-cultivation of Agrobacterium-strain AGL0, harboring plasmid pJFnptII and rye im-mature embryos in liquid medium allowed a high throughput and facilitated washing of the cultures to avoid Agrobacterium overgrowth. Transgenic plants were phenotypically normal and fully fertile, which might be aconsequence of the short time in tissue culture. The selection with paromomycin exclusively during the regen-eration allowed the efficient recovery of transgenic events without interfering with somatic embryogenesis. Southern blot analysis confirmed the independent nature of the analyzed plants and indicated single copy inserts in more than 50% of them. Segregation analysis confirmed single locus integration and stable transgene expression in most of the lines, while one line with multiple locus integration was also observed. The analysis of T-DNA:: plant DNA boundary sequences revealed examples of exclusion of vector sequences, deletion of a few bases of the T-DNA or insertion of up to 29 bases of the vector backbone. This stresses the importance of detailed analysis of the inserted transgenes in order to identify events with the desired integration profile.     

Agrobacterium-mediated transformation of large-flowered purslane (Portulaca grandiflora H.).

Transformation of Portulaca grandiflora has been developed with Agrobacterium tumefaciens strains A281 and T1272. Transformation was assessed by the following criteria: selection of hormone independent callus, antibiotic-resistant callus, and transgenic antibiotic-resistant plants. In addition, DNA hybridization analysis demonstrated that the DNA from tumor lines contained sequences homologous to binary vector T-DNA of strain A281. Following transformation with strain T1272, segregation analysis of the progeny of transgenic plants showed that the transgene was inherited in a Mendelian manner. The kanamycin-resistant progeny tested contained the T-DNA sequence of the strain T1272.     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.     

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.     

农杆菌T-复合物的形成与转运研究进展

在简要介绍农杆菌T-DNA转运全过程的基础上,结合作者近年的工作,重点对T-复合物的形成和T-复合物在农杆菌细胞内的转运机理的最新进展进行归纳和评述．农杆菌能够将其Ti质粒上的一段DNA以单链DNA-蛋白质复合物(简称T-复合物)的形式,通过其细胞两端的四型分泌系统(typeⅣ secretion system,T4SS)转运到宿主植物中,并使宿主发生遗传转化,因而农杆菌介导的T-DNA转运技术已成为应用最广泛的植物转基因技术,同时,由于转运T-复合物的T4SS也是某些质粒接合转移和许多病源微生物分泌致病效应蛋白的通道,因此,农杆菌T-DNA转运机理的研究受到了广泛的重视和关注,使得这方面的研究进展非常迅速．     

胰蛋白酶抑制剂基因siRNA表达体系的构建

以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因KSTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654bp,与已发表的KSTI3基因序列同源性达99%。将反义 正义基因片段插入到pBI121 35S启动子下,构建重组质粒pBIKSTI3。通过冻融法将该重组质粒转入农杆菌EHA105中,获得了siRNA表达体系。利用农杆菌介导法将带有pBIKSTI3的菌株转化大豆,从2棵再生植株中得到2 100bp的特异性扩增条带,而未转化的植株中无该片段的产生。