| The origin and evolution of viruses indicate the direction of virus variation, and new viral variants are always produced during virus evolution. Therefore, understanding viral evolution processes will help us to develop more effective antiviral strategy which avoid large scale virus outbreak. Most plant viruses are RNA genomic, and RNA recombination is the main way for evolution. The phenomenon of RNA recombination occurs in a growing number of viruses. Despite extensive studies, the molecular mechanism of RNA recombination is still not well understood. In this study, Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) which belong to the same genus were used as objects. CMV RNA1,CMV RNA2 and TAV RNA3 were obtained by in vitro transcription, then were mixed to get psedorecombinant viruses and these viruses were inoculated to N.Clevelandii respectively. The RNA recombinantion mechanism was discussed by detecting recombination intermediates and pathogenicity of recombinant virus, the results are as follows:(1) Five infectious clones with T7 promoter used as templets, CMV RNA1, CMV RNA2T2B, CMV RNA2, CMV RNA2W2B,TAV RNA3△163(A) transcripts were obtained by in vitro transcription. Then RNA1, RNA2, RNA3 were mixed to three kinds of pseudorecombinant viruses named C1C2T3△163(A), C1C2T2BT3△163(A), C1C2W2B-T3△163(A) separately, and c2, qt, qw for short.(2) Using the artificial pseudorecombinant viruses c2, qt, qw, N.Clevelandii was infected. Q-CMV was designed as positive contral, while healthy plant was designed as negative contral. Both qt and qw were infectious on N.Clevelandii, while c2 didn't cause any symptoms. qt has more severe symptoms than that of qw, presenting mosaic, dwarfing, dark green in leaves. This shows that the 2b protein from different sources will affect the symptoms of pseudorecombinant viruses.(3) N.Clevelandii was infected by the artificial pseudorecombinant viruses c2, qt, qw. Total RNA of infected leaves at 14 dpi, 28 dpi and 35 dpi was isolated for Northern blot analysis. And the results showed that a new CMV specific band at the little up position of the CMV RNA3 band was observed in 35dpi qt. RT-PCR analysis showed that there were many recombination intermediates between TAVRNA3 and CMV RNA1 in 35dpi qt, and recombination sites were various(4) Sequence analysis revealed that recombination sites are always located in the range of 277nt from 2109nt to 2386nt of the TAVRNA3, and the two RNA sequences near the recombination sites are homologous. 5′-AGGTCCGAAGACGTTAAACTAC-3′( box1), But the recombination sites are not in this homologous sequence, but in the downstream of box1.Obviously, box1 did not play a role in promoting homologous recombination. Among them, the longest recombinant is that the whole TAVRNA3 was connected with the conserved regions 310nt of CMV RNA1. The recombination way which maybe involved a new recombi- nation mechanism. All recombination intermediates were ended with TLS which was high conserved sequence. This indicated CMV replicase might play a key role in viral RNA recombination, for it can recognize the initiation sites for RNA synthesis.(5) Recombinant cloning vectors were constructed, and pseudorecombinant viruses of intermediates were acquired by in vitro transcription. N.Clevelandii was infected by these artificial pseudorecombinant viruses. The symptoms were mottled, vein clearing, dark green, etc. A stable recombinant virus C1C2T2BT3pRE-8 was found by RT-PCR analysis. Viruses were purified from C1C2T2BT3pRE-8, infected N.Clevelandii leaves after four passages. We found that C1C2T2BT3pRE-8 caused apex necrosis in N.glutinosa. Keywords : RNA recombination; artificial pseudorecombinant viruses; in vitro...
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