Preliminary Study On The Effect Of Potato Spindle Tuber Viroid Structural Variation On Replication And Trafficking Function

Potato is the fourth largest food crop in the world.and it is susceptible to virus infection during cultivation.Potato spindle tuber viroid(PSTVd)is a kind of viroid that seriously harms potato production.The current research on the biological characteristics,detection technology and genome sequencing of PSTVd is relatively numerous.but its replication.transportation and pathogenic mechanism are not yet clear.The PSTVd genome contains 27 stem-loop structures with unknown specific function.Based on the particularity of viruses and viroids with simple structures and less RNA molecules,it is a hot topic in current research to use viruses and viroids to study RNA secondary structure and function.In this study,a PSTVd isolaten was collected and detected by RT-PCR technology:different mutants were obtained by closing part of the stem-loop structure of PSTVd.and then in situ hybridization technology and fluorescent quantitative PCR technology were used to understand the trafficking and replication of PSTVd by closing the stem loop structure.In addition,the variation characteristics of PSTVd in tobacco were also analyzed based on high-throughput sequencing technology.The main research contents are as follows:(1)Total RNA was extracted from potato tubers,and a 359 bp specific band was obtained by RT-PCR amplification.After cloning and sequencing,this isolation showed 96.5%-98.1%homology with other PSTVd sequences from GenBank.(2)Tobacco leaves is enzymatically hydrolyzed use enzymatic solution prepared with cellulase and pectinase in the dark,the mixed liquid was filtered through a 400 mesh screen.washed with W5 solution and centrifuged to prepare tobacco protoplasts.Protoplasts were suspended in MMg solution,and the RNA of PSTVd was successfully introduced into tobacco protoplasts by polyethylene glycol method.The total RNA is extracted after one night culture.and the quantitative PCR technology can effectively analyze the replication ability of different PSTVd mutants.Preparation of ten PSTVd mutants with closed different stem-loop structures.Using in vitro transcription technology,the closed-loop mutant plasmid was transcribed into viroid RNA.which was inoculated into tobacco leaves by friction inoculation.After three weeks,frozen sections of the leaf tissue were prepared,then distribution of PSTVd in tobacco tissues was analyzed using,in situ hybridization technique.Combined with the results of in situ hybridization and fluorescent quantitative PCR,it showed that the replication ability of Loop4 was significantly affected and its trafficking ability was destroyed.The replication ability of Loop6 was affected,transshipment ability was affected.The replication ability of Loopl7 and Loop 18 were not significantly affected and their trafficking ability was destroyed.The replication ability of Loop20 was not significantly affected,and the trafficking ability was affected.The replication ability of Loop10.Loop 12.Loop24.Loop25 and Loop26 were affected.and the trafficking capabilities were destroyed.It is speculated that Loop4 is related to replication function:Loop 17.Loop18 and Loop20 are related to trafficking function:Loop6,Loop10.Loop 12.Loop24.Loop25 and Loop26 are related to replication and trafficking function.(3)The PSTVd in 16 single seed sample,which was obtained from tobacco plants infected with PSTVd,was detected by RT-PCR technology,and all samples obtained specific PSTVd bands.The results showed that the seeds form the tobacco plant infected with PSTVd had a positive rate of 100%.The in situ hybridization technique was used to locate PSTVd in the seeds.The results showed that PSTVd was mainly distributed in the seed epidermis and did not enter the embryo.The seeds obtained from the tobacco plants infected with PSTVd were used as materials.and tobacco seedlings were obtained through planting.RT-PCR results showed that all plants were not infected by PSTVd.Combined with RT-PCR and in situ hybridization results,it shows that tobacco seed epidermis can carry PSTVd but PSTVd cannot spread to seedlings through tobacco seeds.(4)Tobacco leaves infected with PSTVd for two months were used as experimental materials to extract total RNA.and high-throughput sequencing technology was used to analyze the variation of PSTVd in tobacco plants.A total of 20 mutation sites were obtained.mainly located in the left terminal region.pathogenic region.the junction of the central conserved region and the variable region of the secondary structure of the PSTVd genome.Mutation types include 5 types of mutations.such as base-undirected mutations.base-directed mutations.base deletions and mutations.base deletions.base insertions and mutations.Among 5 mutation types,base-directed mutation is the most main one,which contains 13 base-directed mutation sites.Most mutations cause changes in the RNA secondar structure of PSTVd.