| Cucumber green mottle mosaic virus(CGMMV),as a new invasive pest,is one of the important viruses that endanger Cucurbitaceae crops in the worldwide.The occurrence and harm of the viral disease became more and more serious in crop production.This virus disease has caused huge economic losses to the gourd crop production in many provinces and cities in China.At present,there is no disease resistant cultivar and high effective disease control method.Therefore,this paper focuses on the pathosystems of CGMMV-cucumber and CGMMV-Nicotiana benthamiana interactions following three aspects:1)screening the optimal reference genes for the normalization of miRNA expression in cucumber under CGMMV stress,providing a reliable basis for the analysis of miRNA expression characteristics.2)digging out miRNAs and their target genes involved in the interactions between cucumber and CGMMV,analyzing thier expression characteristics,exploring the function and regulatory network of cucumber miRNA in response to CGMMV infection,and providing a new perspective for the selection of cucumber resistance gene.3)silencing functional genes of CGMMV by artificial miRNA technology,the CGMMV-resistant N.benthamiana plants were obtained,providing new ideas for the cultivation of disease-resistant varieties.The main findings are as follows:1)Identifying optimal reference genes for the normalization of miRNA expression in cucumber under CGMMV stress.The expression levels of seven reference genes,including Actin,Tubulin,EF-1?,18S rRNA,Ubiquitin,GAPDH and Cyclophilin,were detected at cucumber leaf,stem and root samples that were collected at different days post inoculation with CGMMV using reverse transcription-quantitative polymerase chain reaction(RT-qPCR).The expression stability of seven reference genes was analyzed using delta-Ct,geNorm,NormFinder,and BestKeeper as well as the comparative tool RefFinder.The stability of reference gene was subsequently validated using many miRNAs,for example,miR159.The results showed that EF-1? and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf,root and stem samples,while Ubiquitin and EF-1? were the most suitable combination overall.2)Functional analysis of miRNA responding to CGMMV infection in cucumber.CGMMV-response 47 cucumber miRNA families and 3046 target transcriptions were obtained by degradome sequencing and bioinformatics analysis.The expression characteristics and cleavage sites of miRNAs and their target genes involved in plant-pathogen interactions and plant hormone signal transduction were analyzed and verificated by real-time quantitative PCR,Northern blot and 5'-RLM-RACE.The results shown that the expression levels of 10 miRNAs and 11 target genes had significant differences in leaf samples from 12 h to 15 d after CGMMV infection,among which the expression of some miRNAs were negatively correlated with the expression of their target genes(miR172/XM011656616.1,miR393/NM001280617.1,miR858/XM004140251.2).Most miRNAs cleaved their target mRNAs mainly at the tenth nucleotide of 5'-end of miRNA mature sequence.Moreover,a hypothetical regulatory network linking miRNAs,their target genes and downstream biological processes was proposed to indicate the role of these miRNAs in the cucumber-CGMMV pathosystem.For example,miR159 targets serine/threonine protein kinase PBSl,a positive regulator that responds to biotic and abiotic stresses,csa-mirn6-3p regulates CP32,a calcium-dependent protein kinase,and participates in plant disease resistance.3)Evaluation of artificial miRNA-mediated virus resistance.We constructed six amiRNAs expression vectors,which targeted conserved sequences of CGMMV genes for coat protein(CP),movement protein(MP)or replicase(Rep).Transgenic N.benthamiana plants with overexpression of amiRNA were obtained by Agrobacterium tumefaciens-mediated genetic transformation.The results showed that amiRNA-infiltrated N.benthamiana plants expressing high levels of amiRl-CP,amiR4-MP or amiR6-Rqp exhibited viral 'resistance' or 'tolerance' with reduced viral replication and incidence of CGMMV disease.Plants expressing low level of amiR2-CP,or moderate levels of amiR3-MP and amiR5-Rep remained susceptible to CGMMV and displayed mosaic or mottle disease symptoms.Comprehensive analysis showed that the level of resistance mediated by artificial miRNA targeting CGMMV CP gene was the highest.The above results proved that the application of gene silencing technology mediated by artificial miRNA have protected plants from viral diseases. |
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