ABSTRACT
Full length cDNAs of Cucumber mosaic virus(CMV)CB7 strain,causing necrosis on Nicotiana glutinosa,were obtained by RT-PCR,using viral genomic RNAs as templates.cDNAs of CMV-CB7 genomic RNAs were cloned and sequenced and results indicated that RNA1,2 and 3 was 3 356 nt,3 045 nt and 2 218 nt,respectively(accordingly Accession Number EF216866,DQ785470 and EF216867).Infectious RNA transcripts from cDNA clones of CMV-CB7 were inoculated onto N.glutinosa and the seedlings of host plants displayed necrosis symptom,whist that of CMV-Fny induced typical mosaic symptoms.Through pseudorecombination between CMV-CB7 and CMV-Fny genomic RNAs,the genetic determinant of necrosis phenotype was mapped to RNA2.Chimeric infectious clones consisting of partial sequences of RNA2 derived from CMV-CB7 and CMV-Fny,respectively,were obtained by Overlapping PCR.Pathogenic analysis with those chimeric RNA2 revealed that 2b gene or 3' UTR of CMV-CB7 RNA2 was responsible for the necrotic pathotype.Northern blotting analysis reflected that both necrotic and non-necrotic viruses accumulated to similar levels of genomic RNAs in host plants.Therefore,necrotic phenotype induced on N.glutinosa was not related to the level of accumulation of CMV genomic RNAs.
ABSTRACT
The 2b protein encoded by Cucumber mosaic virus(CMV)plays an important pathogenicity role in many solanaceous hosts,but mechanism of inducing disease is still unknown.In order to investigate virulence of the 2b protein on Nicotiana glutinosa plants,in terms of chloroplast structure and photosynthesis,a mutant Fny-CMV?2bpro,which cannot express the 2b protein,was achieved by introducing mutant sites in the 2b gene of Fny-CMV.N.glutinosa seedlings were inoculated with wild-type Fny-CMV and the mutant Fny-CMV?2bpro,and were analyzed for symptom expression,chlorophyll content,photosynthetic rate,and ultra-structural alteration of chloroplast.Up to 30 days post inoculation,wild-type Fny-CMV caused symptoms of severe mosaic,leaf deformation,and stunting,reduced photosynthetic rate and chlorophyll content,and altered the ultra-structure and morphological characters of the chloroplasts.However,host seedlings inoculated with the mutant Fny-CMV?2bpro expressed only slight mosaic symptom.Their photosynthetic rates and chlorophyll contents were not significantly different from those of the mock-inoculated plants,and the ultra-structure and morphological characters of their chloroplasts appeared to be normal.The observed low photosynthetic rates and chlorophyll contents were related to the breakage of the chloroplast morphology and ultra-structure.Results of Northern blotting showed that the virulence of 2b protein was associated with high accumulation level of CMV progeny RNAs in systemic leaves.Non-expression of the 2b protein reduced the accumulation levels of its genomic RNAs 1 and 2.The level of subgenomic RNA4,encoding CP protein,was found to be significantly decreased.
ABSTRACT
Three purification methods ,based on differential centrifugation,precipitation by polyethylene glycol (PEG)and ultra-speed centrifugation,were compared for purification of an alfalfa mosaic virus (AMVSY)previously isolated from Trifolium repens. The Purified virus was observed under electron microscope, measured,by ultra-violet absorpton analysis and protein determination with SDS-polyacrylamide gel electrophoresis. Our results showed that a method by alternative use of sodium phosphate buffers containing 0. 1mol/L EDTA and 0. 1mol/L MgSO4 achieved the best purification with less miscellaneous protein contamination,integrate virus particles and relatively high yield, which was of 47.6mg virion per hundred grams of fresh leaves of Chanopodium quinoa inoculated with AMV-SY. The coat protein of AMY-SY was tested for about 29 kilo-Dalton.
ABSTRACT
Objective The DNA fingerprints of cultivated Pinellia ternata collected from different geographic regions were generated by using AFLP markers to find the feasibility in analyzing their genetic diversity, relationship, and germplasm identification. Methods The DNA polymorphism of 51 cultivated germplasm of P. ternata collected from 17 different habitats and four cultivars of P. pedatisecta (outgroup) were detected by AFLP molecular markers. Results The DNA fingerprints of 51 individuals of P. ternata were obviously distinguished by eight pairs of high polymorphic and efficient primer combinations screened from 64 primer combinations. The phylogenetic clustering results revealed that all the tested cultivars were fully differentiated, and individuals from the same regions were mainly clustered together. Moreover, cultivars from East-China, including Zhejiang and Jiangsu Provinces, displayed clear genetic distinction from other regions. The clustering results were strongly supported by Bootstrap test. Conclusion AFLP Markers can be potentially used in analyzing of genetic diversity, relationship, and germplasm identification of this medicinal plant, and the germplasm from regions of East-China, including Zhejiang and Jiangsu Provinces, displays the relative separate genetic characters from other regions.