T-DNA Flanking Sequences Research Articles

Transgenic poplar (Populus davidiana×P. bolleana Loucne) expressing dsRNA of insect chitinase gene: lines identification and resistance assay.

Poplar is a valuable tree species that is distributed all over the world. However, many insect pests infest poplar trees and have caused significant damage. To control poplar pests, we transformed a poplar species, Populus davidiana × P. bolleana Loucne, with the dsRNA of the chitinase gene of a poplar defoliator, Clostera anastomosis (Linnaeus) (Lepidoptera: Notodontidae), employing an Agrobaterium-mediated approach. The transgenic plant has been identified by cloning the T-DNA flanking sequences using TAIL-PCR and quantifying the expression of the dsRNA using qPCR. The toxicity assay of the transgenic poplar lines was carried out by feeding the target insect species (C. anastomosis). The results showed that, in C. anastomosis, the activity of chitinase was significantly decreased, consistent with the expression on mRNA levels, and the larval mortality was significantly increased. These results suggested that the transgenic poplar of dsRNA could be used for pest control.

The T-DNA integration characteristics of monokaryotic mutant library in Lentinula edodes

At present, proteomic and transcriptomic analysis are generally used in the study of gene function of edible fungi, and there are few reports of gene function using mutant library. In this study, the Agrobacterium tumefaciens-mediated transformation (ATMT) system of monokaryotic Lentinula edodes was used for constructing a T-DNA insertional mutant library, and over 800 transgenic strains were collected. Forty-eight strains were randomly selected for T-DNA integration characteristic analysis by whole genome resequencing, and the insertion and integration model of T-DNA in L. edodes were presented. These results lay a foundation for gene function research of L. edodes, and can make it better to understand the insertion and integration mode of exogenous T-DNA in basidiomycetes. By screening the mutant library, 38 mutants with altered thermotolerance were screened out from 221 mutants. According the results of whole genome resequencing, the T-DNA flanking sequences in eight thermotolerancechanged mutants were analyzed and annotated. The further studies for these genes disrupted T-DNA insertion would reveal the molecular mechanisms of high temperature response of L. edodes.

Transgenic japonica rice expressing the cry1C gene is resistant to striped stem borers in Northeast China

Rice production and quality are seriously affected by the lepidopteran pest, striped stem borer (SSB), in Northeast China. In this study, a synthetic cry1C gene encoding Bacillus thuringiensis (Bt) δ-endotoxin, which is toxic to lepidopteran pest, was transformed into a japonica rice variety (Jigeng 88) in Northeast China by Agrobacterium-mediated transformation. Through molecular detection and the Basta resistance germination assay, a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1C. Finally, four cry1C-transgenic lines (JL16, JL23, JL41, and JL42) were selected by evaluation of the Cry1C protein level, insect-resistance and agronomic traits. The cry1C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic (NT) control plants. T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1C gene was inserted into the intergenic region of chromosome 11, indicating that its insertion may not interfere with the genes near insertion site. In summary, this study developed four cry1C-transgenic japonica rice lines with high insect resistance and high yield. They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.

Functional Characterization ofPsGPDin Drought Stress Response Using RNA-Seq Analysis of Transgenic Rice Plant

Plants are often exposed to biotic and abiotic stresses that affect plant growth, development, and productivity. Drought is an important abiotic stress that has a particularly serious impact on plant growth and development. We transformed rice with PsGPD using Agrobacterium-mediated transformation. We generated independent PsGPD-homozygous transgenic rice plants selected as single copy/intergenic lines by the TaqMan copy number assay and by T-DNA flanking sequences. These transgenic rice plants showed improvement of drought tolerance compared to wild-type plants under drought condition. RNA sequencing analysis showed that 2,992 genes were transcriptionally affected by the PsGPD transgene or drought treatment. In total, 145 genes were modulated by the PsGPD transgene before and after drought treatment. Among these candidate genes, 4 were up- and downregulated in all four comparisons. Several genes, including Os04t0576900, Os03t0629800, and Os04t0518400 (OsPAL7), were involved in tetrapyrrole synthesis. Os09t0522200 (DREB1A), an important component in hormone signal transduction, is a transcription factor (TF) gene that plays vital roles in stress responses. We partially characterized the functions of PsGPD in the drought stress response and the role of major TFs in the drought tolerance mechanism. These genes will be useful targets for both future research and the breeding of drought tolerance in rice.

Screening of pathogenicity-deficient Fusarium oxysporum mutants established by Agrobacterium tumefaciens-mediated transformation

Apple replant disease (ARD) occurs frequently in old orchards and is an important disease that limits apple production in China. Fusarium oxysporum is one of the causal agents of ARD and its pathogenic mechanism is still unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of F. oxysporum HS2 was constructed via optimized Agrobacterium tumefaciens-mediated transformation (ATMT). The ATMT system yielded 160–200 transformants/106 conidia, indicating a relatively high conversion efficiency. The genetically stable transformants were tested through five generations of successive subculture on hygromycin-free media, and the alien gene was detected via PCR. A total of 3500 transformants were obtained to produce a T-DNA insertion library of F. oxysporum HS2, and 223 of these mutants were randomly selected for sporulation and pathogenicity tests. Most of the tested mutants presented weakened virulence, and one of them displayed obviously attenuated sporulation and reduced pathogenicity. The T-DNA in six pathogenicity-deficient mutants existed as a single copy, as confirmed by Southern blotting. The T-DNA flanking sequence was obtained rapidly by successive chromosome walking via the high-efficiency thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR) method. The T-DNA right flanking sequences of five transformants were identified further, and the obtained sequences were aligned to the genomic sequence, revealing novel loci related to pathogenesis. This study provided a large number of various pathogenesis-related mutants and pathogenesis-related genes, which will be valuable resources for characterizing the molecular mechanisms of pathogenesis-related genes.

Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens-mediated transformation.

Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens-mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B-resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology.

Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3' end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.

Highly efficient generation of T-DNA insertion lines and isolation of flanking sequence tags (FSTs) of Brachypodium distachyon

Brachypodium distachyon (Brachypodium) has been developed as a model system for the temperate grasses. Hence, establishing a large insertion mutant population and identifying T-DNA insertion sites are imperative for functional genomics studies. Currently, Brachypodium has an established T-DNA collection resource, but it is far less than needed. In addition, the conventional methods for the isolation of flanking sequence tags (FSTs) characterizing the T-DNA inserts, such as TAIL-PCR, adapter ligation-mediated PCR, and inverse PCR, are time-consuming, laborious, and expensive. In this study, Agrobacterium-mediated transformation system was optimized to generate T-DNA mutants. Approximately 7000 T-DNA insertion lines were obtained. Furthermore, a simple and highly efficient method was developed to isolate T-DNA flanking sequence tags (FSTs). The procedures simplified the multi-step reaction that is required for the conventional inverse PCR and several reactions were conducted within a microscale reaction system in one tube. It is flexible to isolate FSTs for either individual or large numbers of T-DNA lines. To rapidly process the large-scale sequence data, a serial of Perl scripts were developed using the Perl Programming Language ( http://www.perl.org ). Using these methods, a total of 794 flanking sequences were isolated and analyzed in detail. Although the methods were developed in the process of isolating and analyzing T-DNA flanking sequences of Brachypodium, it can be applied to other plants.

Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi

The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1×106 conidia. Our results indicated that 25°C, acetosyringone concentration of 150μmol/L, and 72h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2×PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

Large-scale phenomics analysis of a T-DNA tagged mutant population.

Rice, Oryza sativa L., is one of the most important crops in the world. With the rising world population, feeding people in a more sustainable and environmentally friendly way becomes increasingly important. Therefore, the rice research community needs to share resources to better understand the functions of rice genes that are the foundation for future agricultural biotechnology development, and one way to achieve this goal is via the extensive study of insertional mutants. We have constructed a large rice insertional mutant population in a japonica rice variety, Tainung 67. The collection contains about 93 000 mutant lines, among them 85% with phenomics data and 65% with flanking sequence data. We screened the phenotypes of 12 individual plants for each line grown under field conditions according to 68 subcategories and 3 quantitative traits. Both phenotypes and integration sites are searchable in the Taiwan Rice Insertional Mutants Database. Detailed analyses of phenomics data, T-DNA flanking sequences, and whole-genome sequencing data for rice insertional mutants can lead to the discovery of novel genes. In addition, studies of mutant phenotypes can reveal relationships among varieties, cultivation locations, and cropping seasons.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis.

Generation of Wheat Transcription Factor FOX Rice Lines and Systematic Screening for Salt and Osmotic Stress Tolerance.

Transcription factors (TFs) play important roles in plant growth, development, and responses to environmental stress. In this study, we collected 1,455 full-length (FL) cDNAs of TFs, representing 45 families, from wheat and its relatives Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum carthlicum, and Triticum aestivum. More than 15,000 T0 TF FOX (Full-length cDNA Over-eXpressing) rice lines were generated; of these, 10,496 lines set seeds. About 14.88% of the T0 plants showed obvious phenotypic changes. T1 lines (5,232 lines) were screened for salt and osmotic stress tolerance using 150 mM NaCl and 20% (v/v) PEG-4000, respectively. Among them, five lines (591, 746, 1647, 1812, and J4065) showed enhanced salt stress tolerance, five lines (591, 746, 898, 1078, and 1647) showed enhanced osmotic stress tolerance, and three lines (591, 746, and 1647) showed both salt and osmotic stress tolerance. Further analysis of the T-DNA flanking sequences showed that line 746 over-expressed TaEREB1, line 898 over-expressed TabZIPD, and lines 1812 and J4065 over-expressed TaOBF1a and TaOBF1b, respectively. The enhanced salt and osmotic stress tolerance of lines 898 and 1812 was confirmed by retransformation of the respective genes. Our results demonstrate that a heterologous FOX system may be used as an alternative genetic resource for the systematic functional analysis of the wheat genome.

The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping.

Enhancer trap systems have been demonstrated to increase the effectiveness of gene identification in rice. In this study, a chlorophyll-deficient mutant, named oscdm1, was screened and characterized in detail from a T-DNA enhancer-tagged population. The oscdm1 plants were different from other chlorophyll-deficient mutants; they produced chlorotic leaves at the third leaf stage, which gradually died with further growth of the plants. However, the oscdm1 plants were able to survive exposure to elevated CO2 levels, similar to photorespiratory mutants. An analysis of the T-DNA flanking sequence in the oscdm1 plants showed that the T-DNA was inserted into the promoter region of a serine hydroxymethyltransferase (SHMT) gene. OsSHMT1 is a key enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for the interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Full-length OsSHMT1 complemented the oscdm1 phenotype, and the downregulation of OsSHMT1 in wild-type plants by RNA interference (RNAi) produced plants that mimicked the oscdm1 phenotype. GUS assays and quantitative PCR revealed the preferential expression of OsSHMT1 in young leaves. TEM revealed serious damage to the thylakoid membrane in oscdm1 chloroplasts. The oscdm1 plants showed more extensive damage than wild type using an IMAGING-PAM fluorometer, especially under high light intensities. OsSHMT1-GFP localized exclusively to mitochondria. Further analysis revealed that the H2O2 content in the oscdm1 plants was twice that in wild type at the fourth leaf stage. This suggests that the thylakoid membrane damage observed in the oscdm1 plants was caused by excessive H2O2. Interestingly, OsSHMT1-overexpressing plants exhibited increased photosynthetic efficiency and improved plant productivity. These results lay the foundation for further study of the OsSHMT1 gene and will help illuminate the functional role of OsSHMT1 in photorespiration in rice.

Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

CmPEX6 , a Gene Involved in Peroxisome Biogenesis, Is Essential for Parasitism and Conidiation by the Sclerotial Parasite Coniothyrium minitans

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6 was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H2O2) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans.

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.

Effort and Contribution of T‐DNA Insertion Mutant Library for Rice Functional Genomics Research in China: Review and Perspective

With the completion of the rice (Oryza sativa L.) genome-sequencing project, the rice research community proposed to characterize the function of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Event-specific qualitative and quantitative detection of transgenic rice Kefeng-8 by characterization of the transgene flanking sequence

The transgenic rice line Kefeng-8 harboring insect-resistant genes Cry1Ac and CpTI showed ideal field performances characterized by high resistance to rice stem borer (Scirpophaga incertulas) and leaf roller (Cnaphalocrocis medinalis). This GM rice line is likely to be approved in China in the near future. In this study, we estimated the insert number of foreign genes, analyzed the flanking sequences of T-DNA in rice genome, and presented an event-specific detection method for this line. The results show that the foreign gene inserted one copy between position 11,774 and 11,805 of chromosome 11 (AC120536.5) in Kefeng-8 genome. Based on these inserts and border sequences, the event-specific qualitative and quantitative PCR system was established for Kefeng-8. The qualitative detection limit was assessed to be 0.1%, and the limit of quantitative method was assessed to be 100 initial template copies. Two mixed rice sample with known Kengfeng-8 contents were used to verify the quantitative method, from which the expected results were observed. This study provides a reliable method and information for detection, identification, and quantification of the presence of GM rice Kefeng-8.

Analysis of Insertion Copy Number and Integration Site of T-DNA in the Genome of Transgenic High Oelic Rapeseed (<i>Brassica napus</i> L.)

In order to acquire the information about the insertion copy number and integration site of T-DNA in the genome of transgenic high oleic acid rapeseed ( Brassica napus L.), we isolated the genomic DNA from the transgenic line W-4, the T2 generation plants, then the transgenic genomic DNA was digested with Bam HI prior to conducting Southern bolt with the probe of a segment of NPT II labeled with Dig. The results showed that only one copy of T-DNA was detected to be integrated into the genome of transgenic line W-4. To isolate the flanking sequence of both right border and left border of T-DNA insertion in the genome, the thermal asymmetric interlaced PCR (TAIL-PCR) was employed by using three or four nested specific primers designed based on the sequence of the vector pCNFIRnos and a short arbitrary degenerate primer (AD1 or AD2) , respectively. The PCR products corresponding to flanking sequences of the right and left border were specifically amplified. The flanking sequence of right border is 470 bp in length which includes a 290 bp genomic sequence and a 180 bp vector sequence based on analysis of VecScreen, while the left border flanking sequence is 641 bp in length including a 365 bp genomic sequence and a 276 bp vector sequence. Further sequence alignment analysis revealed that the 180 bp sequence is identical to the RB border of pCNFIRnos vector, which exists a 62 bp deletion, whereas the later of 276 bp is better identical to the left border of pCNFIRnos vector besides a change from G to A happened. Therefore, it was suggested that the integration of the T-DNA in the genome of transgenic line W-4 should be a kind of vector backbone-free integration. Furthermore, there is no any information about homologous sequence of acquired flanking sequences found in the genome based on the Blastn analysis, which implied that the T-DNA should be integrated into non-coding region of the genome. In conclusion, we considered that the information about the T-DNA insertion copy number, integration site and flanking sequences in the genome of the transgenic rapeseed W-4 might be very useful for the biosafety assessment of genetically modified rapeseed and for the indentification of the transgenic high oleic acid rapeseed.

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.