Development Of Real-time Quantitative PCR(qPCR)detection System And Construction Infectious Clone Of Citrus Yellow Mosaic Virus

Citrus yellow mosaic virus（CYMV）belongs to the order Orterviraless,the family Caulimoviridae,the genus badnavirus.Since it was first discovered in India in 1975,CYMV has spread to citrus growing areas in southern and central India,causing serious hazards to its citrus industry and risking further spread.At present,CYMV has been listed as a subject of quarantine control by countries and regions such as New Zealand,the United States,Japan,and the European Union,but no related studies have been reported in China so far.Therefore,to reduce the biosafety risk of CYMV for citrus industry of China,it is necessary to establish an efficient detection system and explore the properties such as the molecular biology of CYMV.In this study,specific amplification primers were designed based on the conserved sequence of the CYMV coat protein（CP）gene,and a real-time quantitative PCR（qPCR）detection system were established.An infectious clone of CYMV was constructed and subjected to sequence analysis.Then,it was inoculated onto different citrus varieties and herbaceous plants by Agrobacterium-mediated inoculation and symptoms and other biological characteristics were observed.The main findings are as follows:1.Construction of qPCR detection system for CYMVAccording to the conserved sequences of CP gene reported in NCBI,8 pairs of qPCR primers were designed,and the best primer was screened out to be CYMV-q F7/R7.Based on the primer concentration and annealing temperature gradient experiment,it was determined that the optimum concentration was 200 nmol·L^-1,and the optimum annealing temperature was 63℃,and then a qPCR detection system for CYMV was established.The test results showed that the detection system had a good specificity and a sensitivity of 100 times than that of ordinary PCR.Using PCR and the qPCR system established in this paper,360 field citrus samples from different regions of China were detected for CYMV respectively.The results of the two methods were consistent,and no CYMV positive sample were found,excepting for the positive control.Using the qPCR system and citrus sinensis elongation factor 1a as internal reference genes,the contents of CYMV in different parts of sweet orange plant were quantified.The results showed that the CYMV content of symptomatic leaves was higher than that of asymptomatic leaves,and the lateral vein content was higher than that of other parts of leaves.2.Construction of CYMV infectious cloneThe full-length fragment of the CYMV genome was amplified by PCR and connected to the ternary vector p CY by homologous recombination to obtain seven CYMV clones,a ternary shuttle vector that can be replicated in Escherichia coli,yeast and Agrobacterium.A genome-length clone and seven 1.4 times genome-length clones of CYMV were obtained and inoculated onto Eureka lemon（Citrus limon）by Agrobacterium-mediated inoculation.Three and four plants inoculated respectively with CYMV-2 and CYMV-3,both were1.4×genome-length clones,were PCR positive,and showed chlorotic spots.Using DNA of CYMV-3 infected plants as template,PCR was conducted and the expected specific fragment covering the full-length CYMV genome were obtained.When CYMV-3-infected plants were grafted on healthy rough lemon,CYMV-specific bands could be detected by PCR at 90 dpi（days post infection）.CYMV-3 infected leaves were sectioned and observed by electron microscopy.Baculovirus particles with size of 130×30 nm were observed,indicating that CYMV-3 was an infectious clone of CYMV.3.CYMV biological researchCYMV-3 was inoculated onto 10 varieties of citrus by Agrobacterium-mediated vacuum inoculation,and RT-PCR was conducted at 125 dpi,using systemic leaves grown as samples.The results showed that the CYMV positive plants were detected in W.Murcott（C.reticulate×C.sinensis）,No.22 karatachi（Poncirus trifoliata）,Hamlin sweet orange（C.sinensis）,Yuhuanyou（C.grandis）,Banpeiyu pummelo（C.maxima）,Jingchengmiyou（C.maxima）and Wilking mandarin（C.reticulata）.Symptoms observation showed that there was zonal yellowing along the veins on leaves of No.22 karatachi,mild yellow mosaic leaves in Jingchengmiyou,and moderately yellowing leaves in Hamlin sweet orange.CYMV-3 was inoculated onto five citrus varieties（half-year-seedlings）by Agrobacterium-mediated injection inoculation,including Morocco sour orange（C.aurantium）,Rough lemon（C.limonia）,Chandler pummelo（C.maxima）,Duncan grapefruit（C.paradise）,and Madam Vinous orange（C.sinensis）.The infection rate of them by RT-PCR detection at 180 dpi were 85%,90%,68.42%,63.16%and 80%respectively,and the average infection rate was 74%,which was significantly higher than that of using Agrobacterium-mediated vacuum inoculation.Among them,three species showed CYMV infection symptoms:Duncan grapefruit showed chlorotic spots on leaves,the Rough lemon appeared vein flecking,and MV sweet orange appeared mosaic.CYMV-3 was inoculated onto Zea mays,Vigna unguiculata,Sorghum bicolor and Nicotiana benthamiana by Agrobacterium-mediated injection.The RT-PCR results showed that CYMV-3 could infect sorghum and cause mild striped chlorosis,while no infection was detected on Maize,Cowpea and Benthamianaoke.In addition,to understand whether CYMV ORF Ⅰ and ORF II are essential components for the virus infecting,two mutants CYMV 3-1 and CYMV 3-2 were constructed by replaced ORF I and ORF II of CYMV-3 with GFP,respectively.The mutants were inoculated onto sweet orange and Eureka lemon,and subjected to PCR detection at 60 dpi and 120 dpi respectively.The results showed that all the inoculated plants except the control were negative for CYMV,suggesting that ORF I and ORF Ⅱ of CYMV may be necessary for its infection.