Transgenic Wheat Resistant To Barley Yellow Dwarf Virus GPV Using Replicase Gene

Fragments of ORF1 and ORF2 were amplified by RT-PCR after the primers been designed and synthesized according to the results of comparison between partial sequence of GPV and the sequences of other Luteoviruses. The fragment were inserted into pUC 19 plasmid and the recombinants of pUC I 9-ORF 1 and pUC 1 9-ORF2 were obtained. Sequence analysis shows that ORFI contains 1953 nucleotides which can code a protein of 651 amino acids with Mr about 71.4 kD and ORF2 contains 1872 nucleotides which can code a protein of 624 amino acis with Mr about 70.1 kD. There is a long overlape region of 601 nucloetides between ORF1 and ORF2. Compared with other Luteoviruses, there is a highest homhlogy between GPV and RPV. The nucleotide similarity of ORFI is 66.4% and that of ORF2 is 81.7%. Deduced amino acid similarity of P1 is 55.6% and that of P2 is 81.7%. There is a lowest homology between GPV and PAV. The nucleotide similarities of ORF 1 and ORF2 are 44% and 42% respectively and only partial homology. There is almost no homology between deduced amino acid sequence. Based on the sequences of QRFI and ORF2, primers for ORFI and ORF2 were synthesized and the genes were amplified by RT-PCR. Plant expression plasmids of pPPI 12 (containing ORF2) and pPPI 13 (containing ORF 1) were obtained by inserting the fragments into pEmu-mcs-N plasmid. Shan 160 and Longjian 127 were transformed with pPPI 12 and Jinmai 47 and Jingdong 8 were transformed with pPPI 13 respectively via the pollen tube pathway. The seeds harvested were detected by kanamycin screening, resistance assay in fields, dot blot, PCR analysis, and southern blot hybridization. Three positive transgenic lines ofT1 generation were obtained. Two of which were transformed Shan 160 progeny, one was the progeny of transformed Longjian 127. Resistance assay in the greenhouse and in the fields showed that the 2 transgenic lines of Shan 160 have a high lever of resistance to virus infection. When the nontransgenic control showed a serious symptoms, the seedlings of transgenic lines have no symptoms or only slightly yellowing on the leaf apex. The transgenic line of Longjian 127 can delay the symptoms, when the leaves of nontransgenic control were all yellowing the flag leaves of the transgenic line were green or the yellowing area less than 1/3. No positive transgenic plants were found in the progeny transformed with ORF 1. The seeds harvested after transformed with defective ORF2 were detected through kananiycin screening, PCR analysis and southern blot hybridization. 2 positive transgenic lines of Jinniai 47 were obtained out of the 3 varieties-shan 160, Jinmai 47 and Longjian 127ransformed with defective ORF2 gene. The resistance assay in the fields showed that the transgenic line can delay the disease development and had a mild symptoms.