| Objective: Apple Stem Grooving Virus(ASGV) containing positive-sense and single-stranded RNA of 6.5 kb is a type member of betaflexiviridae family and capillovirus genus. ASGV has a broad host range, which can infect pear, apple, apricot, kiwi fruit and so on, causing a serious effect on yield and quality of fruit and industrial productivity. Although thermotherapy coupled with meristem tip culture is the best way to obtain virus-free seedlings, the molecular mechanism that thermotherapy activates certain genes existing in the host expression and some heat shock proteins interacted with virus as well as the transduction and molecular increase pathway of heat shock signal related to disease-resistant reduce the concentration of virus is not clear, giving rise to unstable detoxification effect, which severely restrict effective precaution of virus disease. This study,on the one hand, preliminarily explore the mechanism of heat detoxification by analyzing the expression quantity of disease-resistance and silencing pathway related genes existing in Pyrus pyrifolia under the condition of thermotherapy. On the other hand, this study expects to construct ASGV-Js2 infectious clone which has infected “jinshui no.2” variety of Pyrus pyrifolia to explicit its infection characteristics, thus providing material for further researching the function of antivirus genes in Pyrus pyrifolia.Methods: First, the study equally mixed the total RNA of Pyrus pyrofolia meristem tips which were treated respectively 1days and 5 days at 37℃ and 24℃ to perform RNA-sequencing analysis, then the result was verified by quantitative real-time PCR to further identify differential expression genes involved in diseaseresistant. Second, Pyrus pyrofolia meristem tips infected and not infected by ASGV were treated respectively 1days and 5 days at 37℃ and T24℃, then under the condition of such different treatment, the relative expression quantity of Pp AGO1,Pp AGO2,Pp AGO4,Pp DCL2,Pp DCL4 and Pp Rd Rp1 related to RNA silencing were analyzed in ASGV plantlets and ASGV-free plantlets by q RT-PCR. Third, ASGV infectious clone p MD18-ASGV was obtained by overlap extension PCR and restriction enzyme ligation of nine sequence fragments F1-F9 covering the whole genome of ASGV-Js2. Then infection clone p CB301-ASGV was constructed by the homologous recombination of ASGV and binary vector p CB301-2x35SMCS-HDVRZ-NOS. Finally, the infectivity of ASGV was detected by agrobacterium-mediated infiltration on leaves of Chenopodium quinoa and Nicotiana glutinosa.Results: First, RNA-sequencing result shows that compared to tissue treated at T24, there are 783 differerntially expressed genes including 531 up-regulated genes and 252 down-regulated genes in tissue treated at 37℃. GO function classification shows that these genes associated with biological process, cellular component and molecμlar function, and a large numble of genes are involved in biological process of cellμlar process, metabolic process and response to stimulus. KEGG enrichment analysis shows that genes involved in metabolic pathway and biosynthesis of secondary metabolite are significantly enriched. To verify the differential gene expression result obtained by RNA-sequencing, ten genes including five upregulated genes involved in defense, replicate, translation and five down-regulated genes involved in metabolism, transcription factor and unknown function are randomly chosen in infected plantlets to perform expression analysis of stem tip and basis by real-time PCR. The result shows that expression pattern of ten genes in stem tip is similar to that obtained from RNA-sequencing, which verifies the reliability of RNA-sequencing. Second, the relative expression analysis of differentially expressed genes related to gene silencing pathway: Pp AGO1, Pp AGO2, Pp AGO4, Pp CL2, Pp DCL4 and Pp Rd Rp1 sequence from silencing pathway of Pyrus pyrifolia were firstly cloned and obtained respectively, q RT-PCR analysis showes that Pp AGO1, Pp DCL2, Pp Rd Rp1 expression quantity rise significantly under the condition of thermotherapy, speculating that these genes participate in gene silencing pathway; Pp AGO2 and Pp DCL4 expression are subjected to commen influence of thermotherapy and virus. The content of TRV virus in tobacco decline after inoculation of Pp AGO2 TRV VIGS silence carrier, indicating that Pp AGO2 in Pyrus pyrifolia have an effect on replication and accumulation of virus, but its mechanism is not clear. Third, ASGV-Js2 infectious clone have infectivity by agrobacterium-mediated infection on Chenopodium quinoa and Nicotiana glutinosa as well as subsequent sympotom observation.Conclusions:The study makes clear that the accumulation of virus in Pyrus pyrifolia decline under the condition of thermotherapy, and the differential expression gene Pp899 related to disease resistance is screened under the condition of thermotherapy. The study also clones some gene-silencing related genes Pp AGO1,Pp AGO2,Pp AGO4,Pp DCL2,Pp DCL4 and Pp Rd Rp1 under the condition of hermotherapy and virus infection and analyze expression quantity of these genes, preliminarily speculating that these genes participate in gene silencing pathway by thermotherapy. ASGV infectious clone constructed successfully provide important materials for further function study of genes related to disease-resistant. |
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