| The damage of Peach latent mosaic viroid (PLMVd) is a major factor that causes thelose of the quality and quantity of peach, and the viroid has been considered as one of theseverest systematic pathogen in many countries. But in China, few studies had beencarried out in PLMVd before. In this study, the feasible methods for detection of PLMVdwere developed based on studies of its detection, and the full-length cDNAs of the viroidwere sequenced, and their molecular characteristics were analyzed. Single-strandconformation polymorphism (SSCP) was improved and the molecular composition (i.e.,the population structure) and variability of PLMVd were studed using the developedprotocols, and the molecular mechanism of pathogenic difference of different PLMVdisolates was also analyzed preliminarily. In the end, the polarity of two single strands ofamplified DNA of PLMVd in SSCP gel was identified.(1) Peach samples collected from Wuhan area in spring and summer were subjectedto Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for detection of PLMVd.Results showed that the expected about 337bp products were amplified only from the fivesamples (P1-P5) collected in spring. The samples collected in summer were handled toeliminate contaminated protein and then subjected to RT-PCR again. Results showed thatthe expected amplified products were also obtained from these samples. RT-PCR productsfrom one sample were recovered and biotin labeled for probe preparation. PLMVd of thefive samples was tested and it could be detected by three molecular hybridization waysincluding DNA dot blot, RNA dot blot and tissue printing blot. Among the threehybridization methods, RNA dot blot gave a weakest signal, and tissue printing blot gavea hybridization signal as strong as DNA dot blot. Bi-directional electrophoresis was alsoemployed for detection of PLMVd for a sample, and results revealed the presence aseparate band behind the undivided migrating bands.The distribution of PLMVd in peach was analyzed by tissue printing blot, and strongsignals were observed in tissue printing sites including tender branch, tender fruit, youngleaf with symptom or not, and old leaf with symptom or not, except bark, and resultssuggested that PLMVd distributed in these positions with tiny or no difference of viroidtiter.(2) The full-length genomic RNA of the viroid (named as PLMVd-P3) wassequenced. Results showed that the full-length cDNA of the viroid had a size of 337 nt,which is similar to that of typical PLMVd variants. The nucleotide sequence of the variant had similarities of 90%-96% with other reported ones from different areas. Other isolateswere also cloned and sequenced, and different variants were subjected to analysis of theirbases varibility. Results showed that most of the changes were located in regions, whichincluded both hammerhead structures and loop A and loop B and PSTI arm and othersingle stranded regions. Other regions, delimiting by position 140 and 270 nt, showed lessvariation relatively, including P4 to P10 regions. Analysis of the secondary structures ofthese variants by software revealed a significant diversity of structures. All the structureshad a main core and several stems, stem P1, P9, P10 and P11 were always present in thesestructures and the classical one, whereas stem P2 to P8 folded in various ways and formeddifferent structures with the classical one.(3) Single-strand conformation polymorphism (SSCP) protocol was adopted todevelop sensitive techniques for screening the variability and analyzing the populationstructure of PLMVd. Eight sequenced cDNA clones having changes from 1 to 25nucleotides were chosen as studied materials for SSCP analysis. Results showed that the 8clones gave eight identical profiles in the reported SSCP, which indicated that the existingSSCP had a low sensitivity. Polyacrylamide Gel Electrophoresis (PAGE) conditions,including gel concentration, running voltage, cross-linking ratio of gel and electrophoresistime were evaluated and optimized, and the sensitivity of the classical SSCP protocol wasimproved in different degrees after several conditions changed, and 6 of 8 clones could bedifferentiated from each other. A modified SSCP protocol was developed by combingdifferent gel conditions, and PAGE was made with two equal sections: the upper sectionwas 8% gel with cross-linking of 29:1 and the bottom was 49:1. Results showed that themodified SSCP protocol gave an overall sensitivity in identifying the variability of theseclones. Different SSCP protocols were also applied for analyzing 22 clones from oneisolate again. Results showed that the sensitivity of these protocols used for analyzing the22 clones was similar to that for the 8 clones. To analyze the relationship between thesequences of PLMVd clones and the modified SSCP profiles, no close correlation existedbetween the number of base changes and variation of the modified SSCP band patterns. Itsuggested that the electrophoresis migration of a single DNA was influenced not only bythe amount of varied bases but also by the location of them in the sequence, and the latteracted as a main factor.(4) The molecular varibility of PLMVd within six samples, which showed threemajor symptoms of discoloration, yellowish and mosaic of peach latent mosaic disease onleaves and were collected in different origins, was analyzed by the SSCP protocolsdeveloped. It revealed that PLMVd isolates were composed of a population of genetically related variants (haplotypes), one being predominant with a frequency from 32% to 57%.The proportion of most non-predominant haplotypes was very low and each of them hadonly one clone with a frequency of 4%-5%, but total amount of all non-predominanthaplotypes was high and had a frequency of 43%-68%. Sequences of each isolate wereanalyzed, it showed that the predominant haplotype had very tiny variation with otherhaplotypes within each population, only 1 to 6 bases variation was observed and thesimilarity was 98.5%-99.7% among them. Within each isolate, comparison of thepredominant sequence and the consensus sequence revealed that the predominantsequence was identical with the consensus sequence, which was concluded fromalignment of the known sequences of different haplotypes by bio-software. The resultdemonstrated that the predominant sequence displayed a wide representative for otherswithin a PLMVd population.Similarity and genetic distance of the predominant sequences of the six isolates wereanalyzed. Results indicated that the isolates with same symptoms had higher similaritylevels and smaller genetic distance values from each other, with the similarities more than98.8% and the genetic distance less than 1%. However, the isolates with differentsymptoms had lower similarity levels and higher genetic distance values from each otherrelatively, with the similarities less than 98.5% and the genetic distance more than 1%.The molecular variation characterizations of these isolates were analyzed, and itrevealed that particular variations existed in the isolates showing same symptoms. Theisolates with the symptom of discoloration along leaf sides on its host had a G or U in169nt, and the isolates with the symptom of yellowing had base U and C in 115-116ntrespectively, and the isolates with the symptom of mosaic showed the diversity as (3nt:Del C; 5nt: A and 54nt:U).(5) The polarity of two single strands of amplified cDNA of PLMVd in SSCP gelwas analyzed by asymmetrical PCR-SSCP. Results showed that the single cDNA strandnear the loading well was amplified from minus strand of PLMVd and the one near thegel bottom from the plus one. |
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