Genomic Cloning Of Citrus Tristeza Virus Isolates And Bioinformatical Analysis Of Their Induced MiRNAs In C.sinensis

Citrus tristeza virus (CTV) is one of the most important virus pathogens causing losses in citrusindustry. Mild strain cross protection (MSCP), virus-free propagation materials and resistant CTVcultivars have been adopted to control the CTV disease.MicroRNA (miRNA), an important class of endogenous and non-coding miRNAs with thelength of approximately22nucleotides (nt), has been playing an important role in the regulation ofgene expression, genome epigenetic modification and anti-virus. With the development of modernmolecular biology, new lights have been cast on the fast and effective cultivation of citrus varietiesresisting to CTV and screening of mild CTV strains. The next-generation sequencing technologyoffers an effective approach to identify for identification of miRNAs. Finding out the pathway modeland predicting the interaction mechanism will lay a sound foundation for the development study ofthe miRNA between Citrus sinensis and pathogens.In the study, C. sinensis and CTV were taken as the research objects, Small RNA sequenceslibrary was obtained by high-throughput sequencing technology. With respect to C. sinensis genome,miRNA identification and miRNAs target genes prediction were deployed by the aid ofbioinformatics softwares and relevant databases. The target genes of miRNA were classfied byblasting in the gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) database.The detailed results were shown as follows.1A method was established to detecte CTV by reverse transcription loop-mediated isothermalamplification (RT-LAMP) for the first time. Sensitivity of the RT-LAMP assay was100-fold higherthan a standard RT-PCR method. RT-LAMP can speed up the detection process under routineconditions at a low cost and high accuracy with simple facilities and this can be used as an excellentmethod for CTV detection in both research institutions and rural areas.2The virus-free Toxoptera citricida were obtained by feeding nymphae of T. citricida inscreened greenhouses. Two aphid-transmissible CTV isolates CT11A and CT14A, which had astrong mutual antagonism, were acquired by single aphid transmission.3The complete nucleotide sequence of CT11A and CT14A were acquired by cloning andsequencing. The complete genome size of CT11A strain is19253nt, its ID in GenBank is JQ911664,while the CT14A strain was19247nt in length, and its ID is JQ911663. Phylogenetic analysis of thegenome sequences showed that CT11A genome was closely related to T318A. The full genome andHR region identity were98.89%and99.57%, respectively, but distantly to T36identity which were80.20%and42.14%in both genome and HR region, respectively. CT14A genome was closelyrelated to B165, the complete and HR sequence identity were94.57%and99.64%, respectively. TheCT11A was92.37%in full sequence identity to CT14A, and their HR sequence identity is50.52%.Six clusters strains were classified in accordance with27CTV full sequence cluster alignment. TheCT11A strain belonged to VT clusters, while the CT14A line belonged to B165clusters. Restrictive enzymatic maps of p25and p23genes of CT11A and CT14A strains showed clearly different whendigested by HinfⅠand RsaⅠ. Secondary structure characters of5′-UTR,3′-UTR, HR, p23, p20andp25from the CT11A were not same to those of CT14A.4Data analysis of small RNA library from high throughput sequencing(1) Four distinct small RNA sequences libraries were obtained by high-throughput sequencingtechnology, With respect to C.sinensis genome. A number of conserved and novel miRNA sequenceswere identified. The24nt length small RNA dominated in the small library of C.sinensis free of CTV,while the21nt length small RNA dominated in C. sinensis infected by CTV. The first base of the5′terminal small RNAs perfers―U‖base, some hotspots regions generating small RNAs existed inthe genome of C. sinensis.(2) The expression of miRNAs in four datum sets were analyzed by counting the number oftranscripts per million in libraries.The results showed that99miRNAs were up-regulated in C.sinensis response to CTV infection, while76miRNAs were down-regulated.5Preliminary function analysis of miRNAs(1) A lot of target genes of miRNA were prognosed. Three congruent relationships existingbetween miRNAs and their target genes were revealed: one-to-one, one-to-many and many-to-one.(2) Based on GO (Gene Ontology) database, the target unigenes may be involved in themulticellular organismal process, such as multicellular organismal development transcription,transcription DNA-dependent, RNA biosynthetic process, lignin metabolic process, etc. The targetgenes were showing functions in plant-pathogen interaction, antigen processing and presentation,RNA transport and ribosome biogenesis in eukaryotes pathway, etc in the KEGG database. theplant-pathogen interaction pathway annotation results shown that, there were significant differencesof RAR1, WRKY25, WRKY33, RBOH and CaMCML when C. sinensis was invaded by CTV. Thefacter of CaMCML has a significant difference between CT14A and CT11A invaded the C. sinensis.6The small RNA sequence libraries of CT11A and CT14A were constructed. In these libraries,the22nt length small RNA accounting for45.24%~52.50%of the total sequences was the majority,followed by the21nt length RNA accounting for31.95~35.46%of the total sequences. Somehotspots regions producing small RNAs existed on genes of CTV genome, Small RNAs sequenceswere distributed in an uneven way and mostly clustered on the3′terminal of the CTV genome.