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Dai, Shaotao; Zheng, Ping; Marmey, Philippe; Zhang, Shiping; Tian, Weihua; Chen, Shouyi; Beachy, Roger N.; Fauquet, Claude

doi:10.1023/a:1009687511633

Cited by 270 publications

(45 citation statements)

References 37 publications

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“…Therefore, approximately half of the de novo nupts (8 among 17 events) result from a single ptDNA fragment insertion. The ratio of single to multiple fragments is close to that observed when foreign DNA inserts after biolistics without selection (35) but generally lower than that observed with antibiotic selection (36,37). It is notable that nearly half of the nupts possess multiple inserts at single nuclear loci as is common in biolistic and T-DNA transformation (31,38,39).…”

Section: Insertion Of Single Versus Multiple Ptdna Fragments At Singlsupporting

confidence: 53%

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco

Huang

Ayliffe

Timmis

2004

Proc. Natl. Acad. Sci. U.S.A.

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Transfer of organelle DNA into the nuclear genome has been significant in eukaryotic evolution, because it appears to be the origin of many nuclear genes. Most studies on organelle DNA transfer have been restricted to evolutionary events but experimental systems recently became available to monitor the process in real time. We designed an experimental screen to detect plastid DNA (ptDNA) transfers to the nucleus in whole plants grown under natural conditions. The resultant genotypes facilitated investigation of the evolutionary mechanisms underlying ptDNA transfer and nuclear integration. Here we report the characterization of nuclear loci formed by integration of newly transferred ptDNA. Large, often multiple, fragments of ptDNA between 6.0 and 22.3 kb in size are incorporated into chromosomes at single Mendelian loci. The lack of chloroplast transcripts of comparable size to the ptDNA integrants suggests that DNA molecules are directly involved in the transfer process. Microhomology (2-5 bp) and rearrangements of ptDNA and nuclear DNA were frequently found near integration sites, suggesting that nonhomologous recombination plays a major role in integration. The mechanisms of ptDNA integration appear similar to those of biolistic transformation of plant cells, but no sequence preference was identified near junctions. This article provides substantial molecular analysis of real-time ptDNA transfer and integration that has resulted from natural processes with no involvement of cell injury, infection, and tissue culture. We highlight the impact of cytoplasmic organellar genome mobility on nuclear genome evolution.

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Section: Insertion Of Single Versus Multiple Ptdna Fragments At Singlsupporting

confidence: 53%

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco

Huang

Ayliffe

Timmis

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…BY2 protoplast transfection, Agrobacterium-medicated tobacco transformation, and particle bombardment-mediated transformation of rice were carried out as described (18,20,22).…”

Section: Methodsmentioning

confidence: 99%

RF2b, a rice bZIP transcription activator, interacts with RF2a and is involved in symptom development of rice tungro disease

Dai

Chen

Beachy

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…As a natural plant genetic transformation system, there are some advantages including: full structure of transformed foreign DNA, stable of integration sites, low copy number and less variation of the integrated foreign gene structure, etc. (Brenda et al, 2009;Dai et al, 2001). In contrast, gene bombarding transform technology often leads to multiple-copy integration (Shou et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

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

Chen¹,

Zhang²,

Huang³

et al. 2012

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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 T 2 generation plants, then the transgenic genomic DNA was digested with BamHⅠ prior to conducting Southern bolt with the probe of a segment of NPTⅡ 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.

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Cited by 270 publications

References 37 publications

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco

RF2b, a rice bZIP transcription activator, interacts with RF2a and is involved in symptom development of rice tungro disease

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

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