Nucleotide Sequence Analysis Of The Genome Of Barley Yellow Dwarf Virus GAV And The Movement Protein-mediated Resistance In Transgenic Wheat

Barley yellow dwarf virus-GAV (BYDV-GAV) is a Chinese isolate and serologically related with BYDV-MAV, but they are not identical. Both are transmitted by Sitobion(Macrosiphum) avenae, but GAV is also transmitted by Schizaphis graminum. The complete nucleotide sequence of the genomic RNA of BYDV-GAV was determined and compared with other related viruses. Moreover, through RT-PCR fragments of the gene of movement protein (MP), i.e. ORF4 of genome of GAV, were obtained. Three deletion mutants of the MP gene were cloned into pEmu-mcs-N to construct the plant expression vectors and were transferred into wheat cultivar Yangmai 158 by biolistic particle bombardment. Primary study of the resistance to GAV mediated by MP was carried out.The nucleotide sequence of genome of BYDV-GAV was overlapped by the fragments obtained through RT-PCR .The primers used in RT-PCR were designed according to the conserved sequence among leruoviruses, reported sequences of MAV-PS1 and part sequences of GAV. Sequences of terminal region of genome were identified by 5'RACE and 3' anchored PCR. The genome of BYDV-GAV comprised 5685 nucleotides and has six open reading frames similar to those identified in Australia (PAV-aus). The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of the MAV-PS1 isolate and PAV-aus. Comparison of corresponding ORFs with MAV-PS1 showed that a high degree of identity in that all ORFs showed >90% identities, except ORF5 and ORF6 which had only 87.4% and 70.2% identity, respectively. Comparison between GAV and PAV-aus showed considerable dissimilarity in ORF3 to 6, but un-translated nucleotides at 5'- and 3'-terminal regions shared 88.0% and 80.1% sequence identity respectively. The reported genomic nucleotide sequence of MAV is less than that of GAV, but the comparison of the genomic nucleotide sequence for MAV-PS1 and GAV showed 90.4% sequence identity for the same region of the genome. From sequence comparison, GAV appears to be closely related to MAV and may be a regional isolate of BYDV-MAV.The movement protein (MP) gene of BYDV-GAV was cloned by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. The MP gene has 465nt and encoded 154 amino acids. It shared 97% and 96.8% identity with that of MAV-PS1 in terms of nucleotide and amino acid sequence respectively. The DNA segment was inserted into pGEX-4Tl and expressed in Escherichia coli as a fusion protein toglutathione-S-transferase. The expressed protein was purified by affinity chrornatography and injected into New Zealand white rabbits to produce an antiserum. To test the specificity of the antiserum, immunological detection revealed a 22kD protein could be reacted with the antiserum in the denatured total protein of the infected leaves of oats. Wild type and two deletion mutants of MP gene were cloned into pEmu-mcs-N and constructed the plant expression vector, then transformed into wheat cultivar Yangmai 158 by biolistic particles. Transgenic plants were selected on the medium containing 3-5 mg/L bialaphos. By PCR analysis in TO generation, 14 transgenic plants of MP gene and three transgenic plants of mutant were identified with a transformation frequency of 0.53% and 0.21%, respectively. Through RT-PCR, western blot assay confirmed MP gene was expressed in nine transgenic plants of MP and three transgenic plants of mutant. Primary resistance assay showed that the MP transgenic wheat was more sensitive to the virus infection and showed severe symptoms earlier than the control, while defective MP transgenic lines were resistant to infection. Symptoms were delayed by one month or so. After forty days the flag leaves of the C terminal amino residues-deleted MP transgenic lines only yellowed on the tip of leaves.