Cucumber Mosaic Virus Infecting Pinellia Ternate As A New Araceae Strain

Pinellia ternata is a traditional Chinese medicinal plant which belongs to the Family Araceae. Pinellia ternata basically is a kind of perennial herb. Pinellia ternata sells well at home and abroad market recently and artificial culture of Pinellia ternata was welcomed by the market. However, the yield and quality of the plant was greatly reduced due to lack of culture experience and dependent on wild plants. In addition, severe insect pest and viral diseases damaged the plant production greatly. When wild Pinellia ternata was cultivated extensively and in large scale, pathogens were certain to be a threat to the production. Additionally, the quality declined because of viral infection. We have found that viral infection in Pinellia ternata was related to Cucumber mosaic virus (CMV), however, we know little about its severity and CMV genetic background infecting Pinellia ternate. CMV has been regarded as a model plant virus and the interaction between CMV and its host has been studied extensively. CMV isolated from Pinellia ternata showed quite different characters from other isolates because of its long adaptation with its special host. This thesis constructed complete sequence clones of CMV form Pinellia ternata and identities of its RNA1, RNA2 and RNA3 have been analyzed with other isolates. Especially, the phylogenetic analysis of its CP gene and 3a gene has been studied. Meanwhile, infectious clones of CMV infecting Pinellia ternata have been constructed and its host ranges has been studied by pseudorecombinants reassortment.It is believed to be the first report of CMV complete sequences isolated from Pinellia ternata by RT-PCR and double digestion methods. Phylogenetic analysis revealed that CMV-PGs belonged to subgroup I B. CMV-PGs RNA1 was found consisted of 3336 nucleic acids. The overall sequence identity between CMV-PGs RNA1 and Fny RNA1 was 89.4% while their la sequence similarity was 89.1% at the nucleotide level. The deduced 1a protein identity between CMV-PGs and CMV-Fny was 94.3% at the amino acid level when analyzed by CLUSTAL W. CMV-PGs RNA2 was found consisted of 3037 nucleic acids. The overall sequence identity between CMV-PGs RNA2 and Fny RNA2 was only 87.6% while the 2a sequence similaritywas 87.1% at the nucleotide level. The 2b sequence similarity between CMV-PGs RNA2 and Fny RNA2 was limited to 76.6% at the nucleotide level. The deduced 2a protein and 2b protein identity between CMV-PGs and Fny were 87.3% and 73.9% at the amino acid level respectively aligned by CLUSTAL W.In order to analyze the evolutionary trends of PGs-CMV CP gene and 3a gene more accurately, another CMV-PNb isolated from Pinellia ternata was added to the phylogenetic tree. The analysis results form all of the forty isolates including CMV isolated form Pinellia ternata suggested that at the nucleic acid level, CP genes and 3 a gene obviously formed subgroup IA and subgroup IB. And CMV obtained from P. ternata belonged to subgroup IB when analyzed by CP genes, but the 3 a genes formed another subclade with a high bootstrap. At the amino acid level, although there were no remarkable differences between subgroup IA and subgroup IB for both CP genes and 3 a genes, CP genes and 3 a genes of CMV obtained from P. ternata clustered into 'subgroup \C with a high bootstrap. The results also suggested that CP genes of CMV obtained from P. ternata gained more selection pressure than 3a genes.Infectious clones of PGs-CMV were constructed by RT-PCR method and RNA transcripts were obtained by in vitro transcription. The pseudorecombinants between CMV-PGs and CMV-Fny showed that 14 dpi post inoculation, CMV RNAs were found on the systematic leaves that were inoculated with Fny RNA1 fragment related pseudorecombinants. The molecular identification of RNA3 showed that 7 dpi post inoculation, all the inoculated leaves of N. benthamiana and N. tabacum were found to have RNA3 replication. 10 dpi post inoculation, all the systemic leaves of TV. benthamiana were found to have RNA3 replication. 14 dpi post inoculation, only the systemic leaves of N. benthamiana inoculated with Fny RNA1 fragment related pseudorecombinants were found to have RNA3 replication while again 21 dpi post inoculation, only the systemic leaves of N. benthamiana and N. tabacum inoculated with Fny RNA1 fragment related pseudorecombinants showed RNA3 replication.From the biological and molecular experiments, it could be concluded that Thus, it could be concluded that CMV-PGs couldn't infect N. benthamiana and N. tabacumsystemically only if PGs RNA transcripts with Fny RNAl could infect the tobacco plants systemically. In addition, all the pseudorecombinants between CMV-PGs and CMV-Fny could replicate on inoculated leaves, which showed the activities of RNA transcripts, So RNAl of PGs might be responsible for its inability to infect N. benthamiana and N. tabacum systemically. This might be due to the CMV-PGs's long adaptation to Pinellia ternata and lost the ability to move long distance in the host although it still was able to move form cell to cell. Another reason might be related to the host resistance and made the virus not able to replicate persistently and the virus failed to infect the host systemically.