Construction Of RNAi/PTGS Vectors Of CTV And Transformation In Sour Orange

CTV,a positive-sense single-stranded RNA virus transmitted by aphid and grafting,is a member of the genus Closterovirus of the Closteroviridae.It poses a long term threat to the citrus industry in the world.CTV devastates scion cultivars on sour orange rootstock and imfluences the growth of pomelos,grapefruits and some sweet oranges on other rootstocks.Based on the principle of RNAi/PTGS or RNA silencing technique,this study used the conserved genomic sequences of citrus tristiza virus severe swains to construct 200-400bp inverted repeat cDNAs which would form a hairpin RNA(hpRNA)when expressed.The cDNAs were inserted into the binary vector pCANMIA2301G,under the control of a double 35S promoter.The expression coustructs were transformed into sour orange explants via agrobacteria-mediated transformation.Transgenic plants were regenerated in order to obtain sour orange rootstock with a broad resistance to CTV.Main results:(1)Six complete sequences of CTV severe swains were downloaded form Genebank.Through multiple sequence alignment analysis,conserved fragments in the three RNA silence suppressors (p23,p25 and p20)of CTV were found and primers with restriction sites at their 5'end were thus designed.Three gene fragments were amplified by RT-PCR with the use of the mixed CTV severe strain genomic RNAs as templates and cloned into pTZ57R/T vector.Three clones pTZ57Rp23, pTZ57Rp25,pTZ57Rp20 with the right cloned orientation were obtained by PCR screening of the plasmids using T7 universal primer and the CTV specific primer.Sequence analysis showed that the sequences of the target inverted repeats shared 91.4%to 98.6%of identity with the homologous regions of the 6 severe strains.(2)Nested PCRs were performed using the above three RT-PCR products as templates and seprate primers,designed to carrying a restriction site at the 5'end required for the subsequent assembling of inverted repeat cDNAs,to amplify the fragments close to the 5'end to be used as inverted repeats.The unamplified regions close to the other end of the templates were used as spacers.PCR products were inserted into the constructs obtained in step(1).Restriction analysis was done to confirm the inverted repeats in clones of pTZ57R-hp23,pTZ57R-hp25,pTZ57R-hp20.(3)The GUS gene in the binary vector pCAMBIA2301G was replaced by the inverted repeats obtained through disgestion of pTZ57R-hp23,pTZ57R-hp20 and pTZ57R-hp20 with restriction enzymes and then subcloning of them into the binary plasmid vector pCAMBIA2301G in which the GUS was removed already by restriction.Restriction analysis confirmed that at least two right constructs pCAMBIA2301-ctvhp25 and pCAMBIA2301-ctvhp20 were obtained.Sequence analysis of pTZ57R-hp23 construct showed that a restriction site on its DNA sequence is identical to the chosen cloning site on the binary vector,which resulted in unsuccess in the construction of the binary expression vector due to fregamentation of the construct when digested with the chosen restriction enzyme.The expression constructs containing the inverted repeats of the target sequences p20 and p25 were transformed into agrobacteria EHA105.The pCAMBIA2301-ctvhp25 plasmid-eontaining agrobacterium EHA105-ctvhp25 and the pCAMBIA2301-ctvhp20 plasmid containing bacterium EHA105-ctvhp20 were thus obtained.(4)The Italy sour orange etiolated seedings were cultivated in vitro and cut into segments of 1 cm in length.These segments were infected with EHA105-ctvhp25 and EHA105-ctvhp20 separately. Kanamicin resistant shoots with 3-5 leaves were obtained by co-culturing for 3 days and selective-culturing of the segments for about 3 mouths.Some regenrated shoots were grafted on trifoliate orange rootstock and are growing in greenhouse.The expression of the transgenes in the transgenic plants and the silencing effect on CTV are under evaluation.