The Study On Construction Of Vectors For The Expression Of HpRNA With Cp Gene Of ASGV And The Genetic Transformation Of Tobacco

Pathogen-derived resistance(PDR) is an important strategy for engineered resistance to plant viruses.It is a method of conferring resistance by creating transgenic plants that express genes of a virus in order to create resistance to the virus or related viruses.Most successful and widely application has been the exploitation of plant viral coat protein(CP) genes,thus achieving CP-mediated resistance.Many studies indicated that transcription products of the transformed genes in plants could interact with the related virus RNA and infer its infection or replication.The mechanism of some PDR is involved in post-transcriptional gene silencing(PTGS).Apple stem grooving virus(ASGV),as a +ssRNA virus,is an important virus of apple and pear,which could decrease growth and productivity in infected trees in the latent infection way.The virus could be transmitted into a variety of herbaceous plants through the sap inoculation.Therefore,the indexing on herbaceous hosts is a widely methods for the biologically characterizing the virus.In this study,based on triggering role of dsRNA in RNA silencing the vector expressing a hairpin RNA(hpRNA) from the cp gene of ASGV was constructed and transformed into herbaceous host plant Nicotiana benthamiana by Agrobacterium -mediated method.The object of the study was to provide some useful information for the evaluation of hpRNA induced resistance to homology and other viruses,and to characterize the efficiency of RNA inference.(1) Two pairs of primers were designed based on the sequence of CP gene of ASGV for the amplification of two fragments about 209 bp near 3' terminator and 392 bp near 5 ' terminator.At each end of these primers,two different restriction sites were designed for the later ligation.A clone containing the cp gene of ASGV obtained formerly in our lab was used as template and two fragments about 392bp and 209bp were amplified using these two pairs of primers.The amplified products were sub-cloned into E.coli DH10B.(2) The plasmid containing the 392bp fragment was digested with two sets of restriction enzymes and the target fragment were purified and linked with expression vector pASDS714.The recombined plasmids pASDS714-392(+) and pASDS714-392(-), containing forward and reverse inserts(were transformed into E.coli DH10B again. Furthermore,The plasmid pASDS714-392(+) was digested an other set enzymes and linked with the 392bp fragment obtained by digestion with the same enzymes and the recombinant pASDS714-392(hp) containing forwardly and reversely inserted 392bp fragments at both ends of a intron were obtained. (3) The plasmids pASDS714-209(+) and pASDS714-209(-) containing forwardly or reversely inserted 209bp fragments from 3'terminal of CP gene of ASGV were also constructed by the same way as above.(4) The recombinant plasmids pASDS714-392(-) and pASDS714-392(hp) were transformed into the disces of Nicotiana benthamiana by Agrobacterium-mediated transformation.56 resistant shoots were obtained through,24 transgenic lines of pASDS714-392(-) and 32 transgenic lines of pASDS714-392(hp) were obtained after co-culture and resistance selection.Total DNA was extracted from shoots of resistance lines and detected by PCR with specific primers,and 3 of 6 tested pASDS714-392(-) transformed shoots were positive,all 13 tested pASDS714-392(hp) transformed shoots were positive.