Genome Expressing Of Cucumber Mosaic Virus And Its Relation To Host MicroRNAs

In recent years,the occurrence of virus caused great loss to society and human being, and made them became the new research intersets.Among the prevalent virus,many have (+)RNA genome,which can guide the protein synthesise directly.However,some basic problems of this kind of virus,such as the replication and pathogenicity mechanisms, remain mostly undiscovered.One of the problems is the lack of effective quantification methods to monitor the changes in accumulation of virus RNA during the infection.Cucumber mosaic virus(CMV) is one of the most severe and prevalent plant viruses, and it causes severe yield and quality reduction of crops all over the world.As a typical tripartite RNA virus,it has been long known that the virus has a genome structure consisting of three single-stranded messenger sense RNAs(RNAs 1,2 and 3),and two subgenomic RNAs(RNA 4A and RNA 4),and the functions of CMV replication, movement and pathogenicity have been mapped to each of the five viral RNAs.Therefore, monitor the accumulation kinetics of CMV RNAs in host tissues under different pathogenicity conditions will contribute to better understanding the mechanisms of CMV replication and pathogenicity,and the interaction between environment,virus and host plants.MicroRNAs(miRNAs) are RNA molecules consisting of 20-24 nucleotides that playing key regulatory roles in genes expression.In recent years,it has been demonstrated that miRNAs are important in the process of host-pathogen interaction.However,accurate quantification of the extremely small size of mature miRNAs is a constant technical challenge for traditional approach such as Northern blotting,and the less abundant miRNAs routinely escape detection.Therefore,a more facile and more sensitive method for quantification of miRNAs is required.The rapid developments in technology of molecular biology have provided new tools for solving the above problems.Microarray-based expression profiling and real-time PCR are recently developed powerful technologies for detection and quantification of nucleic acids.With high-throughput ability and high degree of sensitivity,they have greatly benefited many aspects of biological research.Therefore,in this study we focus on further applification of these technologies in different aspects of plant virus research.Concrete contents are as follows:1.A real-time reverse transcript-polymerase chain reaction(RT-PCR) procedure,using the green fluorescent dye SYBR GreenⅠ,was developed for determining the absolute and relative copies of cucumber mosaic virus(CMV) genomic RNAs for the first time. Primers specific to each CMV-Fny ORF were designed and selected through virtual amplification,and the real-time RT-PCR was optimized by varying the concentrations of primers and SYBR GreenⅠchemistry.The standard curve for each ORF was constructed using dilution series of CMV-Fny genome RNAs prepared by in vitro transcription as the standard samples.Using the established quantification system,the copy numbers of genomic RNA 1,RNA 2,RNA 3 and the subgenomic RNA 4 in CMV-Fny virions were quantified,and the ratios were determined to be 1.00:1.17(±0.11):3.58(±0.20):5.81(±0.31).These results were confirmed by Lab-on-a-Chip and Northern blotting assays,and the results of real-time RT-PCR showed higher degree of sensitivity and accuracy.Using 18S rRNA as the internal control,the accumulation kinetics of viral genes in host tissues was examined,and the effects satRNAs,which can change the symptoms induced by helper virus,on accumulation of viral RNAs were determined.2.Optimized the data processing procedure of quantitative real-time RT-PCR.Due to lack of the consideration of real PCR efficiencies in its practical applications,several new mathematical models were used for real-time PCR data processing.Based on the raw fluorescence data,the definitive copy numbers of genomic RNAs in five types of CMV virions(Fny-CMV,FCb7^2b-CMV,FRad35^2b-CMV,FBX^2b-CMV and FNa^2b-CMV) were quantified.Among these mathematical models,the LinReg PCR program demonstrated a higher level of efficiency as considering both the accuracy of results and the procedure of data processing.Moreover,equations were proposed to compare the expression levels of CMV RNAs in virions or in planta.These equations, combined with data analysis by the LinReg PCR program,could significantly enhance the high through-put quantification ability of real-time PCR,and can be used for accurate,reliable and facile investigation of the changes in CMV RNAs accumulation. 3.A plant miRNA microarray(miRNA array) was designed and synthesized,and the response of plant miRNAs to different virus infection was investigated.Since miRNA are highly conserved in related species,a plant miRNA array included 279 unique miRNA was designed and synthesized based on the current plant miRNA information available in database.Using this array,105 unique miRNAs was detected in mock or virus infected tomato leave tissues,and among them 89 miRNAs were found to be differentially expressed after virus infection.Our miRNA microarray results presented herein represent the most comprehensive investigation of the response of tomato miRNAs to virus infection,and provided molecular informations to study the pathogenicity of CMV and the interaction between host plant and pathogen.4.Established a novel plant miRNAs quantification system,using stem-loop reverse transcription followed by SYBR GreenⅠPCR assay.Based on the research of human and animal miRNAs,the novel miRNAs quantification method,stem-loop RT followed by SYBR GreenⅠReal-time PCR assay,was applied to quantify the changes in expression levels of tomato miRNAs and their target mRNAs after virus infection. In compare with mock inoculation,the altered expression levels of both the tested miRNAs and target mRNAs in virus infected tomato seedlings contributes to the hypothesis that plant viruses exploit miRNA pathways during pathogenesis.