The Cloning And Functional Studies Of Giardiavirus MicroRNA

Giardia is a primitive eukaryotes, but also an important zoonosis pathogen has important evolutionary biology research value, as a model organism Giardia there are still many problems to be solved. Giardiavirus by the end of a linear open 7.0 Kb ds RNA and 100 KDa capsid proteins. Small RNA molecules, including si RNA, microRNA, Pi RNA. Where microRNA gene is encoded by an intron or RNA coding intergenic region of the single-stranded RNA molecules of small size of about 18-24 bases, with the target m RNA by base pairing specificity caused by target m RNA degradation or inhibit its translation, and thus gene expression post-transcriptional regulation. At present, for Giardiavirus if there is a microRNA molecules is not clear.In this study, Solexa high-throughput sequencing and bioinformatics analysis, combined VMIR software predicts, initially identified 5 candidate virus Giardia small non-coding RNA, the use of RT-PCR, Northern hybridization, Dicer cleavage, quantitative PCR methods verify that Giardiavirus small non-coding RNA for Giardiavirus microRNA, named GLV microRNA1, its size is 22 nt, located pol protein coding region. This is the first discovered protozoan virus microRNA. The cell location of GLV microRNA1 in giardiavirus infected trophozoites by FISH(Fluorescence in situ hybridization), and the result shows the GLV microRNA1 is the expression in all the trophozoites, which is the same as the giardivirus particles in trophozoites.By knock-down MicroRNA1, quantitative PCR inhibition discovery GLV microRNA1, Giardia viral copy number increased by 30%, indicating that GLV microRNA1 can effectively reduce the number of copies of Giardiavirus. The results of flow cytometry showed that the transfection by GLV microRNA1 will decrease the proportion of green fluorescence positive trophozoites, and the anti-GLV microRNA1 can increase the positive compartment. And the visual observation under the laser scanning confocal microscope showed that, in contrast to the control group, the number of trophozoites with high intensity raised in the anti-GLV microRNA1 group, and the GLV-microRNA1 will reduce the proportion. But for Giardia in terms of morphology, growth aspects curves have not yet found a significant effect. Dual luciferase reporter experiments show that the target was measured gag-pol of 3’UTR GLV microRNA1 play an important role.This is currently the only one in a microRNA gene coding region. The findings may further broaden the way people generate awareness of non-coding RNA, that m RNA may be required between coding and non-coding RNA protein translation there is a balance, this balance may be a new way of regulation of gene expression.