Submitted to: Proceedings of Int'l Association of Plant Tissue Culture and Biotechnology
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2002
Publication Date: 6/17/2002
Citation: Fu, J., Abebe, T., Federico, M.L., Kaeppler, H.F., Skadsen, R.W. 2002. Transformation and expression of an altered antifungal protein hordothionin gene in transgenic barley and oat. Proceedings of Int'l Association of Plant Tissue Culture and Biotechnology. p. 25-A.
Technical Abstract: Alpha-hordothionin is produced in developing barley endosperms and has antimicrobial activity against a wide range of pathogenic bacteria and fungi. We cloned an alpha-hordothionin gene (Hth1) from a cDNA library derived from developing barley endosperms, ligated the nearly full length hordothionin cDNA (Hth1) between the maize ubiquitin promoter and a nos termination sequence in pACH25. The resulting plasmid, Hth1/pAHC, was used to transform barley by particle bombardment of immature embryos. The goal of this project was to express this gene in vegetative tissue to combat fungal infection in barley. However, only an extremely low level of Hth1 mRNA was detected in the leaves of transgenic plants. The observation that deletion of a 5' element in Hth1 dramatically increases the mRNA level of a thioredoxin-hordothionin fusion gene in E. coli pET system prompted us to delete the inhibitory sequence in Hth1 to make a second construct, Hth2/pAHC, for barley transformation. Indeed, the hordothionin mRNA level in the leaves of Hth2 transgenic barley was many times higher than that of Hth1 transformants. The same construct was also used to transform an elite oat cultivar, Belle. Fifteen transgenic oat lines were regenerated from 35 transgenic callus lines and grown in the greenhouse. Negative effects of the transgene Hth2 on transgenic callus growth, regeneration, transgenic plant growth and fertility were observed when compared to those transformed with pAHC25 alone, those that deleted the Hth2 gene and non-transgenic control in both barley and oat.