Studies On Resistance To Potato Virus Y Through DsRNA-mediated Silencing Suppressor

Molecular identification of PVY strains isolated from tobacco and potato plants which derived from different regions in Anhui Province were conducted.Plant expression vector containing inverted repeats fragments of partial HC-Pro gene was constructed,and the construction was transformed into tobacco plants to produce dsRNA.The dsRNA can silence HC-Pro gene of PVY which infected the host,and degradate mRNA of HC-Pro gene and disenable it to express protein.The dsRNA-mediated silencing suppressor HC-Pro gene will elevate RNA silencing efficiency of transgenic tobacco.We will acquire stably inherited transgenic tobacco variety expressing high resistance or immunity to PVY by transforming the construction containing inverted repeats fragments of partial HC-Pro gene into tobacco plants.1.Molecular evolution analysis of CP gene and serological identification of PVY isolates derived from different hosts in Anhui ProvinceTobacco and potato samples infected with PVY were collected from different regions in Anhui Province,and the ELISA results of partial samples are positive. Cloning CP gene of PVY from the sample 4 and 7 was conducted by RT-PCR. Sequencing results indicated that the full length of CP gene of PVY from tobacco (PVY-CP-4) and potato(PVY-CP-7) is 801 nts,and each of them encodes 267 amino acids.A phylogenetic tree based on alignment of CP nucleotide sequences was constructed and the sequence comparing of CP gene was conducted.The results showed that PVY-CP-4 could be grouped into one branch with PVY^O and PVY^N:O and shared the highest sequence similarity(99.4%) with PVY^O(EF026074).It was suggested that PVY-CP-4 derived from tobacco in Hefei should belong to PVY^O. PVY-CP-7 clustered together with PVY^N and PVY^NTN and formed another branch. Furthermore,PVY-CP-7 shared the highest sequence similarity(98.3%) with PVY^NTN (AJ890347),PVY^NTN(EF026075) and PVY^NTN(AJ585342).It was supposed that PVY-CP-7 derived from potato in Wuhe probably belonged to PVY^NTN.2.Cloning and sequence analysis of HC-Pro gene of Potato virus Y(PVY) derived from tobacco plantThe sample 4 was selected for experiment according to the ELISA results. Cloning HC-Pro gene of PVY from the sample 4 was conducted by RT-PCR. Sequencing result indicated that the full length of HC-Pro-4 of PVY is 1359 bp, encoding 453 amino acids.Sequence comparing result showed that sequence of HC-Pro-4 of PVY from tobacco of Anhui Province shared the highest sequence similarity(99.0%) with that of PVY^N(AM268435).Phylogenetic tree was constructed by the HC-Pro-4 of PVY and the other 14 different PVY using the program of the DNAMAN,the tree also indicted that the HC-Pro-4 of PVY had the closest relationship with PVY^N(AM268435).The cloning of HC-Pro gene will benefit the further research work of antiviral genetic engineering.3.Construction of the inverted repeats plant expression vector containing PVYΔHC-Pro(i/r)The specific primers were designed and synthesized according to the published sequence of PVY HC-Pro-4(AM905427).Sense and antisenceΔHC fragment were obtained by PCR amplification.AntisenseΔHC fragment was inserted into vector pSK-In containing sense soybean intron firstly,then the fragment containing intron and antisenseΔHC fragment was digested with restriction enzyme,and subsequently inserted into plant expression vector pBI121 to produce recombinant clone pBI-In-ΔHC-Pro(i).SenseΔHC fragment was inserted into pBI-In-ΔHC-Pro(i) to produce recombinant clone pBI-ΔHC-Pro(r)-In-ΔHC-Pro(i),which contains inverted repeats of PVYΔHC-Pro separated with an intron.4.Agrobacterium-mediated transformation of tobacco plant with inverted repeats recombinant expression vectorpBI-ΔHC-Pro(r)-In-ΔHC-Pro(i) were transformed into A.tumefaciens EHA105 by tri-parental mating method.Transformation of Nicotiana benthamiana and N. tabacum Yunyan 85 was conducted via leaf disc.