| Citrus vein enation virus(CVEV),a member of genus Enamovirus,family Luteoviridae,is a single-stranded RNA virus,causing citrus vein enation disease and woody galls.CVEV has been found in Spain,South Africa,Australia,Brazil,India,the United States,Peru,Turkey and so on.In China,CVEV mainly distributed in Huangyan,Zhejiang province.CVEV can be disseminated by a variety of aphids,so there is epidemic risk of it to some extent.In this paper,a real-time quantitative RT-PCR for CVEV detection was developed,the CVEV full-length cDNA clone was constructed and analysised for infectivity,and the candidategene silencing suppressor of CVEV was identified,so that providing the basis for CVEV epidemiological monitoring,the study of molecular biology and pathogenesis.The main results are summarized as follows: 1.Development of a Quantitative RT-PCR Assay for CVEVA Reverse Transcription Quantitative Polymerase Chain Reaction(RT-qPCR)assay was developed with selective primer pairs(EVq F4/ EVqR4)and optimized conditions.Using the method,only samples infected with Citrus vein enation virus showed positive,while those with other five citrus pathogens were negative.The sensitivity of the method was 100 times higher than the conventional RT-PCR.There was a good linear(R2 = 0.992)relationship between the threshold cycle and CVEV template concentration,while the amplification efficiency of the RT-qPCR was 101.8%.The coefficients of variation of the intra-and inter-assay were both within 2.85%,indicating a good reproducibility of the method.2.Temporal and spatial distribution of CVEV in sour orange plantsThe temporal and spatial distribution of CVEV in sour orange plants was detected by RT-qPCR.The results showed that the titer of CVEV in root was the highest regardless in spring,summer or autumn,followed by that in young bark.Besides,the CVEV titer in young leaf,young bark or old bark was the highest in spring respectively,and showed a declining trend from spring to autumn.3.Construction of CVEV full-length cDNA clone and its sequence analysis36 full-length cDNA clones of CVEV were obtained based on one-step RT-PCR amplification.Sequence alignment results showed that the similarity of the CVEV-XSH with CVEV isolates VE-1 ?pea mosaic virus-1(PEMV-1)and Alfalfa enamovirus-1(AEV)was 98.61% ?90.46% and 90.26%,respectively,suggesting that the CVEV sequence is relatively conserved.At the same time,the phylogenetic tree constructed from full-length sequence showed that CVEV?VE-1 isolates?PEMV-1 and AEV were clustered into one cluster,which was consistent with the results of sequence similarity analysis.4.Infectivity Analysis of CVEV Full-length cDNA clonesAgrobacterium-mediated inoculation method was used to analyze the infectivity of 36 CVEV full-length cDNA clones.But no infectious clones were obtained according to the biological observation and molecular detection.Therefore,a series of tests were designed to explore the factors that affect its infectivity.Firstly,the 5 'end sequence of CVEV was amplified by 5' Race technique and analyzed for its variation.The results showed that the 5 'end sequence of CVEV was highly conserved and no mutation was found.Secondly,The PVX full-length cDNA clone was constructed by the same method as constructing CVEV full-length cDNA clone,and the infectious full-length cDNA of PVX was successfully obtained,which indicated that the binary expression vector p XT1 was effective and the construction method was no problem.Thirdly,in order to avoid the infection failure caused by the instability of recombinant plasmids in E.coli,four positive clones were obtained by transforming agrobacterium directly after connecting CVEV full-length PCR products with expression vector pXT1,but it was failed to screen for infectious clones.Fourthly,Screening of other herbal hosts,but it is regrettable that we failed to screen for herbal hosts of CVEV with toxin leaves extract.In summary,the infection failure of the 36 CVEV clone is not caused by 5' terminal sequence variation,expression vector,construction method,and the instability of recombinant plasmids in E.coli,and further exploration is need.5.Identification of gene silencing suppressor of CVEVIn this study,five open reading frame(ORF)genes of CVEV,including CP gene,were cloned into a binary expression vectors and subjected to identification of gene silencing suppressor by co-inoculating leaves of Nicotiana benthamiana with minireplicon p35mini-BYV.The results showed that the fluorescence intensity of leaves with CP expression was stronger than that of the negative control,but was weaker than that of the positive control,indicating that the CP inhibit gene silencing to some extent.There was no significant difference between the other ORFs and the negative control.The mRNA of GFP in the injected region was quantified by qRT-PCR,and copies of the mRNA in the leaves co-expressed with CP was about double higher than that of negative control and other ORFs,while it was lower than that of positive control.These results suggests that CP may be a weak silencing suppressor... |
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