Identifiction And Analysis Of Small RNAs Infected With Tobacco Mosaic Virus And Cucumber Mosaic Virus In Nicotiana Glutinosa

Plant virus disease spread rapidly and widely in this world.Tobacco mosaic virus(TMV)and Cucumber mosaic virus(CMV)are representative.They have some same characteristics.They are spread widely and difficult to control,and have great damageand variation.In this study,we completed the identification of TMV and CMV and ensured their classified status.Then we inoculated with TMV and CMV in Nicotiana glutinosa,respectively.We identified the small RNAs and its target genes in TMV-Nicotiana glutinosa inaffinity interaction system and CMV-Nicotiana glutinosa affinity interaction system by high-throughput sequencing technology.Moreover,the target genes of miRNA were classified and annotated in the GO and KEGG database.Finally,we studied the defense effects of miR160a and miR398 on TMV and CMV.The main research results were shown as follows:1.We identified the isolates of TMV-Luoyi and CMV-SL and analyzed its nucleic acid sequence.The sequence analysis revealed that RNA1,2 and 3 comprised of 3394,3044 and 2206 nucleotides,respectively.The result of phylogenetic tree showed that CMV-SL strain belongs to the subgroup II strains.There is no recombination between subgroup I and CMV-SL.The isolate showed high homology with subgroup II isolates:97-98%,97-98%and 95-97%within RNA1,RNA2 and RNA3,respectively.The corresponding regions were further compared.The results showed that the coding regions and non-coding regions of CMV-SL had high identity with other subgroup ? strains.The 3'UTR of RNA3 was the most variable than other regions.This suggested that the variation might mostly begin at 3'UTR of RNA3.We amplified TMV CP gene and then analyzed its nucleic acid sequences.The results revealed that TMV CP gene had 480bp nucleotides.The isolations were named TMV-Luoyi.The nucleic acid sequence of TMV-Luoyi showed 85.83%-99.38%homology with other 19 strains.TMV-Luoyi was closely related to TMV-017 and TMV-152 with the identity of 99.38%,but the identity was 85.83%with TMV-Henan.The results suggested that TMV strains existed most variations,which may be associated with host range,different places,etc.2.We identified the small RNA by comparing the high-throughput sequencing datas of uninfected,TMV-Luoyi and CMV-SL infected tobacco.The total original numbers of small RNAs were 26677072,25328238 and 26779823 in the three samples,respectively.The percentage of the Clean reads were 83.18%,69.08%and 83.18%,respectively.The results indicated that there were 415 mRNA,with 209 known miRNA,206 novel miRNA and 39 miRNA families.The number of significantly differencial expressed miRNA between TMV and CMV infected with the control tobacco were 39 and 99,and 14 miRNA of them were verified by fluorescent quantitative PCR.The validation results showed that the results by high-throughput sequencing were believable.Moreover,780 miRNA target genes were predicted.GO functional annotations and enrichment of the target genes and KEGG metabolic pathway analysis of the differencial expressed target genes were taken.The results of all the miRNA target genes in GO showed that the number was 401 in cellular component datas,433 in molecular function datas and 468 in biological process datas.The number of differentially expressed miRNA target genes in biological process was higher than that in cellular component and molecule function infected with TMV and CMV.This result suggested that miRNAs in Nicotiana glutinosa with TMV and CMV were mainly involved in biological processe.The KEGG datas showed that many metabolic pathways were associated with disease resistance in Nicotiana glutinosa infected with viruses.Nicotiana glutinosa with CMV infection involved in more complex defense mechanism against virus infection.3.We analyzed the defense of miR160a and miR398 against TMV-Luoyi and CMV-SL.The research indicated that the accumulation of TMV and CMV could be inhibited by the instantaneous overexpression of miR160a and miR398 in Nicotiana glutinosa,Nicotiana benthamiana and Arabidopsis thaliana,respectively.However,the defense role was different.Next,we detected the transcription level of defense gene.It indicated that miR160a and miR398 mediated different defense signal pathways.For example,miR160a could activate the JA/ET way,but miR398 suppressed this signal way at 8dpi in TMV infection.However,its regulatory mechanism need be further studied.