Molecular Basis For Plant Growth And Development Affected By The Movement Protein Of Barley Yellow Drawf Virus

Barley yellow dwarf virus (BYDVs), representative member of Luteovirus which is transmitted by aphids in the field causes yellow drawf disease in barley, wheat, oats and other cereal crops. The 17 kDa movement protein (MP) has been proved necessary during the spread of virus in plants. Two aspects were maily studied in this thesis:First,the biochemical characteristics of BYDV-MP was studied. After construct ing the recombinant plasmid pET30a-MP and transfering the plasmid into the competent BL21(DE3)pLysS cells, approximately a 20 kDa fusion protein was induced by the addition of isopropyl-β-D-thiogalactoside (IPTG) during cμLtivation . Then two bands, 20 kDa MP and 40kDa homodimer of MP , were detected under Western-blot experiment, which still exited after recovering, purifying and refolding the 20 kDa protein. Accordingly we concluded that MP can dimerize in vitro. Then we did yeast two hybrid experiment to test MP-MP interaction in vivo. Yeast expression vector pGADT7-MP was constructed. After pGADT7-MP and pGBKT7-MP cotransformated AH109, the yeasts were cultivated on the SD/-Leu/-Trp/-Ade/-His , and the cells were found growing well. This proved that MP-MP could interact both in vitro and in vivo.Second, the effect by BYDV-MP on plant growth and development was studied with transgenic technology. MP can localize in the nuclear envelope because of fourα-helix in N-Terminus, and has non-specific ssRNA binding activity with C-Terminus rich in arginine. In order to testify the key functional domains of MP, the primers of MP, NMP(1-250bp), CMP(250-500bp) were synthesized. The recombinant plant expression vectors with 35S strong promoter were constructed and were transferred into Agrobacterium GV3101. Transgene Arabidopsis thaliana with the method of Floral-dip. The seeds harvested were analyzed by PCR. The status of plant growth and development of different transgenic plants were analyzed statistically and the resμLt is that transgeneic plant with GFP:MP and CMP delayed flowering about ten days than Wild type. CDC48 of Arabidopsis is highly expressed in elongation cells and growing points of buds,roots and flowers , so we spectulated that GFP:MP and CMP may affect the CDC genes. We analyzed the gene CDC48 in transgenic plants through Real-time quantitative PCR and found the expression of CDC48 decreased in transgenic plant of gene GFP:MP and CMP. Growth experiment of CDC48 Arabidopsis T-DNA insertion mutants (cdc48) demonstrated that the inhibition or expression reduction of CDC48 induced Arabidopsis growth retardation.This further suggested that MP may affect plant growth and development by inhibiting the expression of CDC48.