Studies On The Specificity Of Aphid Transmission Following Double Infection By GPV And GAV Of BYDV

Barley yellow dwarf virus (BYDV) is the most important virus disease of cereal crops worldwide. To develop and plant resistant cultivars is one of the best ways for controlling the disease, but no natural resistance genes have been identified in oat, barley or wheat so far. In recent years, To transfer foreign genes, especially the viral coat protein (CP) gene into the genome of crops to develop resistance against BYDV was the most common strategy. Several transgenic lines showing resistance to BYDV-GPV have been developed in China. However, questions concerning the potential ecological impact of CP-mediated Virus-resistant transgenic plants have been raised, such as Heterocapsidation, RNA recombination. The aim of this study is to make clear the aphid vector specificity following double infection with GPV and GAV and study molecular mechanism of interaction between GPV and GAV. Technologies developed in the study are also expected to be used as a possible model of evaluation system for biological security of transgenic wheat against virus.In general, GPV is transmitted by Rhopalosiphumpadi Z,.(Rp), but not by Sitobion avenae F. (Ma) and GAV is transmitted by Ma, but not by Rp. In our experiments, the seedlings of Coast-black oats were inoculated with GPV and GAV of BYDV, by means of Rp and Ma, respectively. Among the 93 mixing infected plants, 16 plants altered aphid vector specificity. The data suggested that the heterocapsidation occurred in some plants mixed infection with GPV and GAV, and phenotypic mixing occurred in high proportion in these cases.The progenies of mixing infection were tested by DAS-ELISA with MAV-antiserum. The results showed that the 69 plants of mixing infection , whether the infected plants transmitted by RP or Ma> have very strong reaction to MAV-antiserum. The data supported that that the heterocapsidation occurred in some materials of mixing infection.Based on the CP and RTF gene nucleotide sequence of GAV, GPV and PAV, six pairs of specific primer were designed. 12 of the progenies of mixing infection with GPV and GAV were tested by RT-PCR. The CP gene were obtained from all of 12 samples with the specific primer of GAV-CP, but no products with the specific primer of GPV-CP. The fragment of RTP gene were amplified from 6 of 12 samples with the specific primers of GAV-RTP, no product was obtained with the specific primer of GPV-RTP. The GAV-CP gene could be amplified from the total RNA of infected plants transmitted by Rp, so transcapsidation between GPV and GAV of BYDV was supposed. Sequence alignments showed that unusual sequence was not existed in the tested samples and the identity among the sequences was over 99%. The results suggested that there was no evidence to RNA recombination in the mixing infection materials.In addition, the DNA fragments of GAV, GPV and PAV of CP gene amplified by RT-PCR were cloned into the plasmid pGEM-T Easy, which were used to produce DIG-labeled probe for Nucleic acid spot hybridization(NASH) technique.