An affinity-based genome walking method to find transgene integration loci in transgenic genome.

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.

Molecular characterization and expression of a gene encoding cytosolic Hsp90 from Pennisetum glaucum and its role in abiotic stress adaptation.

Heat shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. They have a crucial role in the folding of a set of proteins involved in the regulation of many essential cellular pathways and also re-folding of stress-denatured polypeptides. However, their exact function is still not clearly elucidated. In this study the full-length cDNA encoding for Hsp90 polypeptide and its corresponding gene was isolated from Pennisetum glaucum (designated PgHsp90). PgHsp90 cDNA encoded for a polypeptide of 698 amino acids with a predicted molecular mass of 80.3kDa and shared a high sequence homology (97-81%) to other plant cytosolic Hsp90s and shared less sequence homology (40-45%) to organelle and endoplasmic reticulum specific Hsp90 isoforms. A deduced amino acid sequence possessed three structural domains: N-terminus (1-211) ATP binding domain, middle (281-540) client protein interacting domain and C-terminus (541-698) dimerization domain; the N-terminus and middle domain is linked by a charged linker domain (212-280). It possesses the five-conserved amino acid signature sequence motifs characteristic of the Hsp90 family and a C-terminus MEEVD penta-peptide characteristic of the cytosolic Hsp90 isoform. The predicted quaternary architecture generated for PgHsp90 through molecular modeling was globally akin to that of yeast Hsp90. The PgHsp90 gene consists of 3 exons and 2 introns. The position and phasing of these introns were conserved in other plant cytosolic Hsp90 genes. Recombinant PgHsp90 protein was expressed in E. coli and purified to homogeneity, which possessed in vitro chaperone activity. E. coli expressing PgHsp90 protein showed enhanced tolerance to heat, salt and dehydration stresses. The quantitative up-regulation of PgHsp90 gene expression positively correlates in response to different stresses to meet the additional demand for protein folding support. Cumulatively, the in vivo and in vitro experiments indicated that PgHsp90 plays an adaptive or protective role to counter the stress induced protein damage.