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Titolo:
Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders
Autore:
Zheng, SJ; Henken, B; Sofiari, E; Jacobsen, E; Krens, FA; Kik, C;
Indirizzi:
Univ Wageningen & Res Ctr, Plant Res Int, NL-6700 AA Wageningen, Netherlands Univ Wageningen & Res Ctr Wageningen Netherlands NL-6700 AA Netherlands AARD, RIV, Lembang, Indonesia AARD Lembang IndonesiaAARD, RIV, Lembang, Indonesia Univ Wageningen & Res Ctr, Dept Plant Sci, NL-6700 AJ Wageningen, Netherlands Univ Wageningen & Res Ctr Wageningen Netherlands NL-6700 AJ Netherlands
Titolo Testata:
TRANSGENIC RESEARCH
fascicolo: 3, volume: 10, anno: 2001,
pagine: 237 - 245
SICI:
0962-8819(200106)10:3<237:MCOTS(>2.0.ZU;2-0
Fonte:
ISI
Lingua:
ENG
Soggetto:
POLYMERASE CHAIN-REACTION; MEDIATED TRANSFORMATION; ARABIDOPSIS-THALIANA; PLASMID RESCUE; AMPLIFICATION; WALKING; PLANTS; GENE; JUNCTIONS; FRAGMENTS;
Keywords:
adaptor ligation PCR; Allium cepa; copy number; genomic target site; T-DNA integration;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
36
Recensione:
Indirizzi per estratti:
Indirizzo: Kik, C Univ Wageningen & Res Ctr, Plant Res Int, POB 16, NL-6700 AA Wageningen, Netherlands Univ Wageningen & Res Ctr POB 16 Wageningen Netherlands NL-6700 AA
Citazione:
S.J. Zheng et al., "Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders", TRANSGEN RE, 10(3), 2001, pp. 237-245

Abstract

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 patternsobtained 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 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomicDNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 02/04/20 alle ore 12:14:08