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Detailed Southern analysis of selected transgenic lines

Molecular characterisation of transgenic materials by Southern blot analysis was conducted.

Genomic DNA was isolated from leaves of transgenic or parental plants. Approximately 10 µg aliquots of genomic DNA were subjected to digestion with appropriate restriction enzymes, separated on agarose gel, blotted and fixed onto nylon membranes. DNA was hybridised with 32P or non-radioactively labelled DNA probes.

For PHA-E expressing rice, Southern hybridisation of transformants demonstrated the presence of three insertions in the selected transgenic line. This result was obtained using both PHA-E coding DNA and the ubiquitin promoter as DNA probes.

Southern blot analysis of the genomic DNA from GNA expressing plants indicated that there was only one insertion event of the transgene in the rice genome. A genomic library was constructed, using a Lambda vector, to identify the site of insertion. Screening of the library with radioactively labelled gna coding DNA revealed that the transgene was integrated into a non protein coding region of the rice genome.

For Bt expressing rice, an internal fragment of the gus (uidA) gene and a cry1Ab fragment generated by PCR were used as probes for hybridisation. Results from Southern analyses clearly showed that the transgene was inserted at a single locus. Inverse PCR was also employed in an attempt to characterise the site of insertion of the transformation cassette. Bt expressing rice was transformed via Agrobacterium T-DNA, thus IPCR primers were designed based on the sequences of the left and right borders of the vector. The generated PCR product of approx 1.2 KB was cloned into a sequencing vector. After sequencing, Similarity studies using BLASTX or BLASTN indicated that transposition of the transgene occurred within a retrotransposable element-coding region in the rice genome.

Furthermore, the results indicate that both single and multiple insertion events may occur when particle bombardment is used to transform plants.

More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

Contacto

Angharad GATEHOUSE, (Director of Research, School of Biology)
Tel.: +44-191-2464811
Fax: +44-191-2225228
Correo electrónico
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