Study On Construction And Identification As Well As Function Of RNA Interference Lentiviral Vectors Of Inhibin Gene In Cattle And Buffalo

Inhibin is a kind of glycoprotein hormones produced by the ovarian granulose cells and testis Sertoli cells. A disulfide bond linksα-andβ-subunits to form the heterodimer inhibin and a subunits is the necessary element for inhibin to carry on its function.Inhibin has negative feedback on FSH Synthesis and secretion, that effect animal's reproduction function. On female, it can inhibit follicular development and promote granulocyte apoptosis. On male, it can inhibit spermatogenesis process.Therefore, using RNAi technology to down-regulate of the inhibin level can promote follicular development to increase the number of large follicle, and improve the production of sperm. However, there are no reports on whether interfering the inhibin synthesis and secretion totally could influence the animal reproduction and other physiological functions maintaining.In the research, we use lentiviral vector-RNAi (Lv-RNAi) technology to inhibit the expression of cattle INHA gene and buffalo INHA gene respectively.We choose the INHA genes of cattle and buffalo as the target gene to design shRNA for RNAi, containing shRNAa, shRNAb, shRNAc, shRNAd, shRNAH1, shRNAy and shRNAs.Lentiviral vectors Va, Vb, Vc, Vd, VH1, Vy, Vs expressing shRNAa, shRNAb, shRNAc, shRNAd, shRNAH1, shRNAy, shRNAs respectively are constructed by gateway clone technology in 3 steps.Lentiviral vectors transfected cattle and buffalo ovarian granulose cells respectively, and then extracted total RNA for real time PCR to analysis the relative expression of cattle INHA gene and buffalo INHA gene.Analyze the effect of the lentiviral vectors on inhibiting target gene expression according to the relative expression of target genes in every group.1,Lentiviral vectors used in cattle have different efficiency of inhibiting INHA gene expressionWe get that Va, Vb, Vc, Vd, VH1 have a significant effect on inhibiting cattle INHA gene expression respectively. Va, Vb have high efficiency on inhibiting cattle INHA gene expression, Va, Vc, Vd have moderate efficiency, Lvd, VH1 have low efficiency.2,Lentiviral vectors used in buffalo have different efficiency of inhibiting INHA gene expressionVa, Vb, Vc, Vd, VH1 have a significant effect on inhibiting buffalo INHA gene expression respectively. Va, Vb, Vd have a high efficiency on inhibiting buffalo INHA gene, Vc, Vshui have low efficiency.3,Different combinations of vectors have different efficiency of inhibiting INHA gene expressionUsing the different lentivirus groups to interfere the target gene, we get that Y group have no significant effect on inhibiting cattle INHA gene expression, though E, F, H, J, N have significant effect.4,RNAi efficiency mediated by plasmid DNA vector-LvdVd, Lvd transfected cattle ovarian granulose cells respectively, then, analyzed the effect on target gene expression. The result Vd has higher interference effect than Lvd show that lentiviral vector is better than plasmid vector in mediating RNAi.5,Long term expression of GFP in 293FT cellsGreen fluorescence can be still observed with unreduced intensity and the normal cell morphology, when the 293 FT cells transfected by VGFP subculture to 39 day.Conclusion:Va, Vb, Vc, Vd can effectively inhibit both cattle and buffalo INHA gene expression. As the target fragments have high homology and nearer position, as the vectors have similar effects. Different groups have different effects. When the two vectors have similar shRNA, both of their interference abilities are depressed. When the two vectors have different shRNA, both of their interference abilities are improved. In the first kind group, if adding a vector containing different shRNA, all of their ability were improved, and when the shRNA target gene positions were more far and ability were improved more. However, when the group contains four more vectors, all of the abilities were more depressed. When the target sequence is at the end of the target genes, even though only 7 bp match, interference can also occur. Lentiviral vector is better than plasmid vector in mediating RNAi. Long term expression of GFP mediated via VGFP in 293FT cells show that lentiviral vectors packaging in this study can insert the foreign gene into the host chromosome. Therefore, the foreign gene can be expressed and inherited to offspring cells stably as well as constantly.