Integration Loci Structure And Genetic Expression Of Green Fluorescent Protein (gfp) Gene In Transgenic Barley (Horsdeum Vulgare L.)

The key for transgenic plants could be used widely is the stable heredity and expression of transgene. Four doubled haploid barley lines, named as A,C,D and F, respectively, were derived from microspores by particle bombardment. These four lines with different expression level of gfp gene were studied for the expression level of gfp and the gfp location in plant genome. The integration sites were detected by FISH using the gfp plasmid DNA as a probe. The results were line A,C,D and F all had a single insert site on chromosome 7 at different location, while line F has a second insert site on chromosome 5. Line D was silent transgene of gfp. Line F showed strong expression of gfp on its root tips. The different expression of gfp between D and F may be resulted from the physical location of the gfp in plant genome.Line F was intercrossed with A, C, D and CK(control line), respectively. In order to explore the heredity of transgene gfp expressed in transgenic barley lines, all the parental lines, F1 and F2 generations of different intercrossing groups were measured for gfp expression level on root tips and pollen. The results were 8 groups (line F intercrossed with A, C, D and CK) had the same expression level of fluorescence on root tips and pollen. It indicated that the gfp transgene was completely nucleus heredity. Most intercrossed lines (6/8) except two crosses were stable heritability on root tips and pollen. The results were consistent with Mendelian genetic law.To more fully characterize the internal structure of transgenic loci, line D and line F which were transformed by the same plasmid were analyzed by Southern Blot and plasmid rescue. The results of Southern Blot demonstrated that the two lines both had a fragment which had the same size as the transformed plasmid. Using different restriction endonuclease, plasmid rescue were performed, and several rescue plasmids were obtained. They owned the same size as the transformation plasmid and contained the same gfp gene identified by PCR. Sequence results of these rescue plasmids exhibited that most of them had the same sequence as the transformed plasmid except one composed of short truncated sequences.All results above proved that transgene in line D and line F were direct head-to-tail repeats arrangement of transformed plasmid. The similar characteristics of transgenes in both transgenic barley lines implicated similar mechanisms of transgenic integration and arrangement regardless of the detail number of co-transformed plasmids.