Differential Expression Of MicroRNAs Between Distinct Genotypes Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular protozoan, infects both humansand animals. Apart from causing significant economic losses in animal husbandry, T.gondii also causes toxoplasmosis in humans. It is asymptomatic in most chronicinfections, but during the acute infection, it can cause severe disease, such as damagesin eyes, brains or other body organs in immunocompromized hosts. According to thedistinct genotypes, T. gondii was divided into three predominant clonal lineages; theclonal lineages also differ in a number of phenotypes such as growth rate, efficiencyin migration and transmigration in tissues, which was presents as diverse virlence.The mechanisms that cause the differences between various virlence of the strain of T.gondii are still unclear enough.RNAs are one of the most important biomolecules to organism, small noncodingRNAs are a member of them, which act as an endogenous regulate factor in cells,including microRNA (miRNA),small interfering RNA (siRNA), endogenous siRNA(esiRNA) and piwi-interacting RNA (piRNA), all of them form a complex networkwhich regulate gene expression at the posttranscriptional level. MicroRNAs playimportant roles in organisms development, cell proliferation, differentiation, apoptosisand metabolism. They regulate gene expression by programming the RNA inducedsilencing complex (miRISC) which interacts with the complementary sequences ofmRNAs causing their translational inhibition or cleavage. MiRNA expression hasbeen shown to be tissue-specific as well as temporally regulated during exploremiRNA potential function in many species. So far there is rarely research onmicroRNAs of T. gondii, more efforts on its miRNA will make contribution to studythe virlence of the parasite and the relationship between parasite and host. We aimedat small noncoding RNA between different genotypes of T. gondii, focusing on themiRNA which presents specific expression and differential expression. To survey miRNAs in T. gondii，two small RNA libraries of T. gondii of RH(genotypeⅠ) and ME49(genotypeⅡ) strains were constructed and sequenced usingthe Solexa sequencing technology. A total of1,083,320sequence reads were obtainedfrom both two libraries, in total,339novel miRNAs and17conserved miRNAscomprised of2miRNA families were found. There were lots of miRNA which wasspecific expression and differential expression；besides，there was a clear difference inthe tendency of the length in small RNAs between the two different genotype strains；Mature miRNA derived from different arms in a same precursor between two distinctstrains. In addition, the proportions of small ncRNAs in the two libraries (ME49andRH) were different, which was studied by employing bioinformatics analysis.To confirm the Solexa sequencing result, real-time PCR method, Cloning andSequencing, Northern blot was employed. The5’ digoxin-labeled, LNA-modifiedoligonucleotide probe was used to hybridization with5miRNAs, which was strain-specific expressed in two different genotypes of T. gondii. Results of Northern blotwere in accordance with the Solexa sequencing results. Due to the existence of theprecursors of mature miRNA in T. gondii, it was impossible to get effectivequantitative results between mature miRNA with different expression, even so,cloning and sequencing assisted in reconfirming the precursor sequences in T. gondii,agreed with the result of Northern blot.In summary，we have conducted a systematic analysis of microRNAs in twostrains of T. gondii of distinct genotypes by combination of Solexa high throughputsequencing and bioinformatics analysis. Both strain-specific and commonly expressedmicroRNAs with various expression levels were identified. In general, T. gondiipossesses a unique profile of microRNAs which is phylogentically distinct from otherorganisms. Futher, we observed strain-specific microRNAs can be generated from thesame pre-microRNA by arm-switching. It is postulated that microRNAs are linked tothe shift of parasite virulence. The data of this study have paved a new way for futureresearch gene regulation in T. gondii.