The Instability Of Geminivirus TYLCCNV Infectious Clone In Agrobacterium

Geminivirus are a class of plant virus that have single-strand circular DNA.Plants show different symptoms infected by geminivirus,including curly,yellow leave and late-blooming.The tandem repeat viral infectious clone was constructed and inoculated into plants by Agrobacterium to facilitate the research operation.Tomato yellow leaf curl China virus(TYLCCNV)is studied in our laboratory.It was found that the onset of viral symptoms was delayed or even less susceptible to disease after infecting the plants with the preservation strain.We found that complete viral infectious clone could not be detected in Agrobacterium through preliminary analysis.We reconstructed the complete viral infectious clone to take research on the instability of the infectious clone and the relevant influence factors,offering the reference for the viral infectious vector research.The main findings are as follows:1.The complete TYLCCNV-Y10 A infectious vector was transformed into Agrobacterium,extracting the plasmids of the overnight culture and transforming E.coli.The fifty percent of sixty mono-clones which were chose from the medium plate have lacked.Detecting revealed that the missing fragment of the plasmids was a viral replication unit and most of the coding regions of replication-association protein Rep/AC1 and AC4 were deficient in the remaining sequences.2.After continuous subculture,the detectable complete clones were reduced,even nothing.Ninety percent of vectors remained stable when the Y10 A infectious vector was transformed into AGL1 Agrobacterium(rec A background,HR defecting),illustrating the instability of viral infectious vectors is connected with homologous recombination.3.According to the gene coding region of AC1 and AC4,we constructed Y10 Amini vector and the mutation vectors of AC1 and AC4,transforming it into Agrobacterium to explore whether AC1 or AC4 affects the instability of viral infectious clone.The results showed that Y10 Amini was unstable unless AC1 mutates,which indicates that the instability of viral infectious clones was related to AC1.4.To investigate the effects of DNA breakage and homologous recombination on the stability of vectors,we constructed DNA breakage models based on I-Sce I and I-Sce I K223 I.The results indicate that breaking repair through homologous recombination may result in weak deletion in repeat regions.5.To investigate the effect of the duplication of the viral replication origin(ori)on the instability of vector,we constructed the lacking clone and simulated clone of Y10A-ori and transformed into Agrobacterium.The ori deficient clone remains stable,but the Y10A-ori simulation vector experienced instability due to the presence of AC1,indicating that the duplication of the ori region leads to the instability.6.Beet curly top virus(BCTV)was another geminivirus we study,whose infectious clone is stable in Agrobacterium and serious disease condition infected plants.In conclusion,this study has found that the infectious clone of geminivirus is unstable in Agrobacterium,which makes the onset time delay and viral symptom of inoculated plants inconsistency.The instability of TYLCCNV infectious clone is connected with homologous recombination and cased by viral replication-associated protein Rep/AC1 and ori repeat.The instability of infectious clone of different geminivirus is difference in Agrobacterium,which maybe be connected with the activity of Rep/AC1.Results of our experiment offer reference for the geminivirus infectious clone infected plant stably.