Wheat transformation with HVA1 gene by microprojectile bombardment

Master of ScienceGenetics Interdepartmental ProgramTo improve drought tolerance, a gene encoding a late embryogenesis abundant (LEA) protein, HVAJ, from barley (Hordeum vulgare L.) was introduced into winter wheat Jagger (hard red winter wheat) and Lakin (hard white winter wheat). The gene construction, containing HVA1 gene driven by the rice Actl promoter and the selectable marker gene, bar, under the control of CaMV35S promoter, was delivered to wheat embryogenic calli by means of microprojectile bombardment. One transgenic wheat plant of Jagger was obtained in this study. This plant survived in medium containing 5 mg/1 ammonium glufosinate during the tissue culture processes and has normal morphology. The plant tested positive for the PCR analysis of bar gene and was resistant to 0.1% (v/v) herbicide LibertyTM. Southern blot analysis showed the integration of bar and HVA1 gene into the genome of this plant. The 27 kDa of HVA1 protein also was detected in this plant as revealed by Western blot analysis

Functional characterization of a Baculovirus fibroblast growth factor

Doctor of PhilosophyDepartment of BiologyA. Lorena PassarelliBaculoviridae is the only known virus family that encodes genes with homology to vertebrate and invertebrate fibroblast growth factors (fgfs), key regulators of developmental processes affecting cell growth, differentiation, and motility. The role of viral fgfs during infection is not known. In this study, we investigated gene regulation and function of the Autographa californica M nucleopolyhedrovirus (AcMNPV) fgf during infection of permissive insect cells. We demonstrated that the AcMNPV fgf, vfgf, was transcribed as a 0.6-kb mRNA at early times post infection, but as part of a 1.4-kb bicistronic mRNA at late times. To determine its function, we examined common characteristics between vFGF and other well-characterized FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate chemokinesis of different types of insect cells. We also constructed a recombinant of AcMNPV lacking a functional vfgf and analyzed it in two insect cell lines. The kinetics of budded virus production were similar in the parental and vfgf-deficient viruses in two cell lines and at both high and low multiplicities of infection. In addition, we observed no obvious differences in the viral DNA synthesis and the protein kinetic profiles of cells infected with the mutant and parental viruses. Finally, coinfection of vfgf-containing and -deficient viruses and their passage for several generations did not reveal a consistent growth advantage for either virus. We propose that vFGF is the signal that directs the motility of uninfected tracheal or blood cells to infected tissues, enabling the virus to infect additional cells and spread systemically in the insect host. This proposal may explain a dispensable role for vfgf during virus infection in cell culture; nonetheless, we expect a distinct phenotypic difference between vfgf-deficient and vfgf-containing viruses during infection in the insect host