Studies On Virus Identification Research And Aconite Mosaic Disease Of Aconitum Carmichaelii Debx

Aconite,also known as Aconitum or Fuzi tablets,Radix Aconiti Lateralis Preparata is the sub-root of Aconitum carmichaelii Debx.It has many effects such as strong heart,analgesic,anti-inflammatory,local anesthetic,and anti-tumor,anti-aging,It can lower blood sugar,and rehabilitate the patient.The main chemical components are aconitine,mesaconitine,hypaconitine,deoxyaconitine,tauroaconine and so on.tuber propagation is generally used,but this vegetative propagation method can accumulate pests and diseases,leading to a decline in yield and quality degradation.Aconite infests a large number of pests and causes many diseases including:white peony disease,powdery mildew,root-knot nematode disease,mosaic disease,etc.At the same time,continuous cropping obstacles accumulate aconite disease,resulting in serious sensibility,degeneration of aconite varieties and severely affect normal growth has greatly limited the aconite planting production.In recent years,Aconitum mosaic virus disease has occurred in Jiangyou where is the the local production area of Aconite.The incidence of high-level or field plots can reach 70%to 80%.In this study,we identified the pathogens and their characteristics of the Aconitum mosaic virus plant in Jiangyou by reverse transcription polymerase chain reaction?RT-PCR?,and analyzed the differences in the expression levels of the virus in different parts of the plant.Lays a theoretical foundation for the prevention and treatment of aconite mosaic disease.The results are as follows:1.Cloning and Sequence Analysis of CMV Coat Protein Gene in AconitumSpecific primers:CMV?cp-F,R?were designed based on a 375-bp conserved sequence of the coat protein gene of CMV that was included in GenBank.RT-PCR amplification was performed.Reverse transcription of cDNA from Aconitum leaves and fibrous roots was used as a substrate.Negative controls were selected as leaves of healthy tissue culture Aconitum.The optimal cloning conditions were optimized with the annealing temperature of 55.7°C.After purification,transformation and sequencing,a nucleotide sequence of 375 bp in length was obtained.The nucleotide and amino acid sequence identities of the cp gene from 18 representative CMV isolates at home and abroad were 74.8%-98.2%and 73.4%-99.2%,and the nucleotide similarity to the CMV subgroup II was 96.1%-98.2%,amino acid similarity with CMV subgroup II is96.0%-99.2%.The highest amino acid sequence similarity between CMV isolates and Chinese isolates?CMV-PCT2?was 99.2%.Genetic evolution analysis showed that closely related to Chinese isolates?Zin and Hubei-1?and American isolates?DKRD?,Gathered in the CMV subgroup II branch and named CMV-FZ.2.Analysis of physicochemical properties of CMV coat protein gene in Mosaic disease AconitumThe physicochemical properties of the protein were predicted and analyzed.It is assumed that the molecular formula is C₆₁₀H₁₀₀₄N₁₈₆O₁₈₃S₃,the relative molecular weight is 13,952 Da,and the isoelectric point?PI?is 10.77.The protein has an instability coefficient of 62.80 and is an unstable protein.The fat coefficient was 71.76and the hydrophilicity coefficient was-0.650.The CMV-FZ gene encodes 125 amino acids and compares the amino acid composition of the protein.The most two amino acids are Ser?serine?and Arg?arginine?,and the proportions are 12.0%and 11.2%,respectively.There are 10 negatively charged amino acid residues?Asp+Glu?and 23positively charged amino acid residues?Arg+Lys?.Hydrophobicity predictions indicate that the CMV-FZ gene is a hydrophilic protein.The predicted protein sequence CMV-FZ subcellular localization in the chloroplast transit peptide and the nucleus,Transmembrane region predictions indicate that the protein is a non-transmembrane protein,signal peptide predicts that the protein may play a signal recognition role in transmembrane transport,mature peptide Beginning with the 17th amino acid,containing the signal peptide sequence ALMDKSESTNASRTSRR,the cleavage site is located at amino acids 17-18.3.Protein structure prediction and motif prediction of CMV coat protein gene in Mosaic disease AconitumThe secondary structure prediction analysis of the protein encoded by CMV-FZ gene showed that there were 5??Strand,e?folds in the protein secondary structure,the content is 17.1%,and the content of a Helix?h?is 15.4%.The maximum content of Coil?c?is 67.5%.The prediction of protein tertiary structure was performed on CMV-FZ,and the prediction result was very similar to the reported 1f15.1 protein,1f15.1 being CMV?Cucumber mosaic virus CMV??STRAIN FNY?.The conserved motif analysis was performed on the cp protein sequences of CMV-FZ and other 4 CMV isolates and PSV,respectively.The results showed that these CMV amino acid sequences contain highly conserved motif 1-motif 6 core conserved motifs.The conserved motifs of the CMV-FZ protein and the CMV-Wem and Hubei-1 proteins belong to CMV subgroup II are almost identical.It is further explained that CMV-FZ belongs to CMV subgroup II.4.Quantitative detection of CMV virus expression in different tissues of AconitumClone the cDNAs of the samples with the reference primers EF1 and ACTIN,and sequence them.According to the gene alignment analysis,The similarity of the EF1gene of?FJ389748.1?EF1 included in NCBI was 93%.And in the Aconitum root,fibrous roots,leaves expressed relatively stable.The EF1 gene is relatively conserved.The EF1 internal reference gene was selected as the Aconitum internal reference gene.Real-time fluorescence quantitative detection of leaves,tuberous roots,and fibrous roots was performed.The differences in the expression of CMV-FZ in various parts of Aconitum were analyzed.The results showed that the expression levels of CMV in leaves,fibrous roots,and tubers were significant differences,of which the highest expression level in the leaves,the second fibrous root,the least root.In summary,through the molecular identification of the plant pathogens of Aconitum in Sichuan Jiangyou where is the local production area of Aconite,and the characteristics of the gene sequence analysis,the results showed that Aconitum with mosaic symptoms is infested with Cucumber Mosaic Virus.Molecular identification and sequence analysis of Pathogens of Aconitum with mosaic symptoms was carried out,and an identification system for cucumber mosaic virus in Aconitum was obtained to provide a scientific basis for its comprehensive prevention and control.