Detection Of Latent Virus In ’Sai Wai Hong’ Apple And Proteomic Study Of ACLSV Infection In Nicotiana Occidentalis

Apple is our economic fruit tree,long history of cultivation.In recent years,apple virus has become more and more serious,which has caused a lot of problems.Apple stem grooving virus(ASGV),Apple stem pitting virus(ASPV)and Apple chlorotic leaf spot virus(ACLSV)are three latent and recessive viruses that are most common in cultivation of apple trees.In the early stage of this study,we detected the latent virus of’Sai Wai Hong’ apples in Tongliao city by RT-PCR technology,which laid a foundation for the occurrence and prevention of virus disease in apples in Tongliao City.Later,the response mechanism of ACLSV infection in western tobacco was analyzed based on proteomics technology by injecting ACLSV infecting cloned bacteria solution constructed in the laboratory,and the differential proteins in the process of ACLSV infection were screened out in order to find the key specific proteins related to ACLSV.The main findings are as follows:(1)After RT-PCR,the detection rate of ACLSV was the highest in the branch samples of ’Sai Wai Hong’ apple outside the Sea’,up to 30.2%.The ASGV detection rate is the second highest,reaching 22.0%.The detection rate of ASPV was 12.5%.After statistical analysis of the detection rate,the detection rate of ACLSV and ASPV in Kailu Town of Kailu County reached the highest level,among which the detection rate of ACLSV reached 66.7%,the detection rate of ASGV reached 58.3%,and the detection rate of ASPV in Dongfeng town reached 41.7%.The detection rate of three latent recessive viruses was 4.2%,that of ACLSV and ASPV was 4.2%,that of ACLSV and ASGV was 13.5%,and that of ASPV and ASGV was 3.1%.(2)Through this study,we extracted 29 ACLSV isolates from ’Sai Wai Hong’ apple(Gen Bank entry number: ON001687～ON001715),their nucleotide sequence consistency was between 78.1% and 99.9%,and the nucleotide sequence consistency with 15 known ACLSV isolates was also between 69.1% and 93.6%.In addition,by amplification technique,Genetic sequences of the replicating and moving proteins of these isolates were combined to form phylogenetic trees.44 ACLSV isolates were found to be distributed in 7 branches,namely JB,MO-5,Balatonl,P863,group 1,B6 and Ta Tao 5.(3)In this study,we obtained 12 ASPV isolates from ’Sai Wai Hong’ apples(Gen Bank entry number: OL953372～OL953383),their nucleoside sequences were83.6%～99.7% consistent with the nucleoside sequences of 15 known ASPV isolates,and75.4%～91.5% consistent with nucleoside sequences of 15 known ASPV isolates.In addition,we also used some shell protein genes and 3 ’non-coding region sequences.Phylogenetic trees were constructed to better understand the characteristics of the species.Twenty-seven ASPV isolates were distributed on 6 branches,and the isolates of’Sai Wai Hong’ apple were distributed in three different groups Ⅰ,Ⅳ and Ⅴ.(4)In this study,21 ASGV isolates(Gen Bank entry number OL960733～OL960753)were extracted from ’Sai Wai Hong’ apple,and their nucleoglycine sequence consistency was 96.4%～100.0%.Compared with 38 known isolates,Their nucleoglycine sequences were 89.9% to 98.9% consistent,and phylogenetic analysis showed that the 59 ASGV isolates clustered into eight large clades.The ASGV isolate forms a separate branch in the "red outside" apple,known as Group Ⅰ.(5)The proteome of Acls V-infected and healthy tobacco leaves was sequenced.The sequencing results showed that 7967 proteomes could be identified from NCBI library with FDR<1%.Using statistical techniques,we found 780 different levels of gene expression.We used screening criteria of p-value < 0.05,fold change < 0.83 or fold change > 1.2 to determine the expression levels of these genes.Compared with the control,there were 271 up-regulated genes and 509 down-regulated genes.(6)In this experiment,through the GO annotation of 780 differential proteins,the results showed that a total of 53 GO terms were significantly enriched,and the participation of differential proteins in molecular functions involved 22 different biological pathways;The involvement of differential proteins in cell components involves 20 different biological pathways.The involvement of differential proteins in biological function involves 11 different biological pathways.Due to the particularity of species,there were no enrichment results in KEGG Pathway in this study.Through the analysis of the related pathways,it was concluded that the ion stress and osmotic stress induced by ACLSV infection could affect the biosynthesis of other secondary metabolites,resulting in the up-regulation of the co-factors of biosynthesis and the metabolic pathways of vitamin,and the down-regulation of the metabolic pathways of terpenoids and polyketones.ACLSV infection may lead to increased ion and osmotic pressure,changes that affect the metabolism of nucleic acids,cofactors and vitamins,which in turn affect the reduction of carbohydrate metabolic pathways.Under ionic stress and osmotic stress caused by ACLSV infection,large subunits L4,L14,L22,L32 and small subunits S3,S4,S8,S11,S15,S16,S18 of ribosomal proteins were up-regulated.The PPI network was constructed to understand the protein interaction relationship.The contents of RPS4,rpl14,rps12-A and PPA were all high,and the correlation coefficient of interaction was about 1.3.Rpl14,rps12-A,rps15,rps3 and rps7 have a maximum of 7correlation relationships,which indicates that they are very close to each other.