Simple Identification of Transgenic<i>Arabidopsis</i>Plants Carrying a Single Copy of the Integrated Gene

Kihara, Tomonori; Zhao, Changjiu; Kobayashi, Yuriko; Takita, Eiji; Kawazu, Tetsu; Koyama, Hiroyuki

doi:10.1271/bbb.50687

Cited by 33 publications

(23 citation statements)

References 9 publications

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“…Real-time polymerase chain reaction (PCR)-based methodology for the determination of transposase-gene copy number was introduced and demonstrated [34]–[36]. Absolute quantification determines the exact copy number of transposase gene by relating the Ct value to a standard curve.…”

Section: Resultsmentioning

confidence: 99%

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Scanning of Transposable Elements and Analyzing Expression of Transposase Genes of Sweet Potato [Ipomoea batatas]

Ling

Xiang

et al. 2014

PLoS ONE

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BackgroundTransposable elements (TEs) are the most abundant genomic components in eukaryotes and affect the genome by their replications and movements to generate genetic plasticity. Sweet potato performs asexual reproduction generally and the TEs may be an important genetic factor for genome reorganization. Complete identification of TEs is essential for the study of genome evolution. However, the TEs of sweet potato are still poorly understood because of its complex hexaploid genome and difficulty in genome sequencing. The recent availability of the sweet potato transcriptome databases provides an opportunity for discovering and characterizing the expressed TEs.Methodology/Principal FindingsWe first established the integrated-transcriptome database by de novo assembling four published sweet potato transcriptome databases from three cultivars in China. Using sequence-similarity search and analysis, a total of 1,405 TEs including 883 retrotransposons and 522 DNA transposons were predicted and categorized. Depending on mapping sets of RNA-Seq raw short reads to the predicted TEs, we compared the quantities, classifications and expression activities of TEs inter- and intra-cultivars. Moreover, the differential expressions of TEs in seven tissues of Xushu 18 cultivar were analyzed by using Illumina digital gene expression (DGE) tag profiling. It was found that 417 TEs were expressed in one or more tissues and 107 in all seven tissues. Furthermore, the copy number of 11 transposase genes was determined to be 1–3 copies in the genome of sweet potato by Real-time PCR-based absolute quantification.Conclusions/SignificanceOur result provides a new method for TE searching on species with transcriptome sequences while lacking genome information. The searching, identification and expression analysis of TEs will provide useful TE information in sweet potato, which are valuable for the further studies of TE-mediated gene mutation and optimization in asexual reproduction. It contributes to elucidating the roles of TEs in genome evolution.

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Section: Resultsmentioning

confidence: 99%

“…A dilution series is prepared from the cloned plasmids to provide a measure of absolute standard abundance to generate a standard curve. Linearized plasmid was quantified using a spectrophotometer and copy number was calculated for all standards by the following formula [34]–[36]: …”

Section: Methodsmentioning

confidence: 99%

Scanning of Transposable Elements and Analyzing Expression of Transposase Genes of Sweet Potato [Ipomoea batatas]

Ling

Xiang

et al. 2014

PLoS ONE

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“…T3 seeds were used for organic acid excretion assays, growth assays, and transcript analyses. Integration of T-DNA was confirmed by genomic PCR as described previously (Kihara et al, 2006).…”

Section: Stop1-suppressing Tobacco Transgenic Linesmentioning

confidence: 99%

Characterization of AtSTOP1 Orthologous Genes in Tobacco and Other Plant Species

et al. 2013

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Aluminum (Al) and proton (H + ) tolerances are essential traits for plants to adapt to acid soil environments. In Arabidopsis (Arabidopsis thaliana), these tolerances are mediated by a zinc-finger transcription factor, SENSITIVE TO PROTON RHIZOTOXICITY1 (AtSTOP1), which regulates the transcription of multiple genes critical for tolerance to both stressors. Here, the functions of orthologous proteins (STOP1-like proteins) in other plant species were characterized by reverse genetics analyses and in planta complementation assays. RNA interference of a gene for NtSTOP1 repressed Al and H + tolerances of tobacco (Nicotiana tabacum) roots. Tobacco roots released citrate in response to Al, concomitant with the up-regulated transcription of an ortholog of an Al tolerance gene encoding a citratetransporting multidrug and toxic compound extrusion protein. The RNA interference repression of NtSTOP1 blocked this process and also repressed the transcription of another orthologous gene for Al tolerance, ALUMINUM SENSITIVE3, which encodes a prokaryotetype transporter. These results demonstrated that NtSTOP1 regulates Al tolerance in tobacco through the transcriptional regulation of these genes. The in planta complementation assays revealed that other plant species, including woody plants, a legume, and a moss (Physcomitrella patens), possess functional STOP1-like proteins that can activate several H + and Al-tolerance genes in Arabidopsis. Knocking out the gene encoding the STOP1-like protein decreased the Al tolerance of P. patens. Together, our results strongly suggest that transcriptional regulation by STOP1-like proteins is evolutionarily conserved among land plants and that it confers the ability to survive in acid soils through the transcriptional regulation of Al-and H + -tolerance genes.

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“…Expression of the PetC (photosynthetic electron transfer c; At4g03280) served as a quantitative reference. 11 Reactions were performed separately and pooled before loading on to a gel to facilitate analysis. The PCR products of the size expected for the cDNA corresponding to the WT transcript (1522, 1155 and 145 bp) were obtained for all plants indicating the presence of the WT transcript in the homozygous SALK_135513 line (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intronic T-DNA insertion in ArabidopsisNBR1conditionally affects wild-type transcript level

Wawrzyńska

Sirko²

2014

Plant Signaling & Behavior

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The SALK_135513 line of Arabidopsis thaliana is annotated by GenBank to have the T-DNA insertion in the fourth exon of NBR1 (At4g24690). Careful molecular analyses of the homozygous plants of SALK_135513 line indicated the place of T-DNA insertion in the fourth intron. Unexpectedly, 2 kinds of NBR1 transcripts, the wild-type and the mutated, resulting from alternative splicing events, were detected in those plants. Our findings explain the problems encountered by us with phenotypic evaluation of this line and emphasize the necessity for independent verification of the exact insertion site followed by careful expression studies when working with Arabidopsis T-DNA insertional mutants.

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Simple Identification of TransgenicArabidopsisPlants Carrying a Single Copy of the Integrated Gene

Cited by 33 publications

References 9 publications

Scanning of Transposable Elements and Analyzing Expression of Transposase Genes of Sweet Potato [Ipomoea batatas]

Scanning of Transposable Elements and Analyzing Expression of Transposase Genes of Sweet Potato [Ipomoea batatas]

Characterization of AtSTOP1 Orthologous Genes in Tobacco and Other Plant Species

Intronic T-DNA insertion in ArabidopsisNBR1conditionally affects wild-type transcript level

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