RNA干扰不同类型TLR基因对罗氏沼虾免疫相关基因表达的影响

Effects of RNA interferences with different Toll-like receptors on the expression of immune-related genes in Macrobrachium rosenbergii

通过分别注射0.4、0.8和1.2μg/g(siRNA/体质量)小干扰RNA(short-interfering RNAs,siRNA)来比较不同剂量siRNA对罗氏沼虾(Macrobrachium rosenbergii)MrTLR1、MrTLR2和MrTLR3等3种TLR基因表达的抑制效果。荧光定量PCR检测结果显示1.2μg/g组为最适干扰剂量。注射MrTLR1-siRNA 24 h以及48 h后罗氏沼虾血淋巴中髓样分化因子88基因(MyD88)、甲壳素基因(Crustin)和抗脂多糖因子基因(ALF)的表达量相对于注射前均显著下降,同时MrTLR2在鳃中的相对表达量上升;在注射MrTLR2-siRNA后,MyD88以及Crustin在血淋巴中的相对表达量明显下降;而在注射MrTLR3-siRNA后,免疫缺陷同系物基因(IMD)在鳃中的相对表达量显著上升,同时ALF在血淋巴中的相对表达量显著上升。此外,3种TLR基因的沉默对酚氧化酶原基因(proPO)的表达没有影响。研究结果表明:MrTLR1和MrTLR2可调控MyD88以及抗菌肽的表达;而MrTLR3不仅参与Toll信号通路调控,同时也可能影响IMD信号通路。

RNA interference assays of three types of MrTLRs in Macrobrachium rosenbergii were conducted using specific short-interfering RNAs(siRNAs) with doses of 0.4, 0.8, and 1.2 μg/g, and the relative expression levels of three Toll-like receptor genes in gills were analyzed with relative quantitative real-time PCR. The result showed that 1.2 μg/g was the optimal dose of siRNA for RNA interference assay of MrTLRs in the present study. In the RNA interference assay of MrTLR1, the expression of Myeloid differentiation factor 88(MyD88), Crustin and anti-lipopolysaccharide factor(ALF) in MrTLR1-silenced prawns obviously declined at 24 h and 48 h post injection, whereas the MrTLR2 expression was significantly upregulated. Furthermore, the expression levels of MyD88 and Crustin significantly decreased in MrTLR2-silenced prawns, and the silencing of MrTLR3 induced remarkable upregulation of the expression of immune deficiency(IMD) and ALF in M. rosenbergii. Additionally, there was no significant difference of prophenoloxidase(proPO) expression between MrTLRs-silenced groups and scrambled-siRNA group. These results suggested that MrTLR1 and MrTLR2 might be involved in the regulation of antibacterial peptides expression, and MrTLR3 exhibited regulatory effects on the Toll signaling pathway as well as IMD signaling pathway in M. rosenbergii.