家蝇微生物感染对RNA干扰GNBP3基因的作用

Effect of microorganism infection of Musca domestica on RNA interference of GNBP3 gene

为了研究家蝇Musca domestica微生物感染对RNA干扰GNBP3基因的作用,评价微生物感染后对下游抗菌肽水平的影响,本研究用RNA干扰技术沉默家蝇GNBP3基因探索最佳沉默时间及效果,检测RNA干扰后家蝇幼虫存活及化蛹情况,通过微生物喂食途径感染,采用实时荧光定量PCR方法检测抗菌肽基因(cecropins、defensin、diptericin)的表达情况。结果显示注射dsGNBP3后家蝇GNBP3基因在24 h达到有效沉默,沉默GNBP3基因后家蝇幼虫的化蛹率及存活率降低。RNA干扰GNBP3基因后,在未感染组中,cecropins的表达量显著性降低;白色念珠菌感染后,cecropins的表达与未感染组呈相似的趋势(P<0.0001);而大肠杆菌和金黄色葡萄球菌感染后,cecropins的表达量显著提高(P<0.05)。对家蝇diptericin基因的表达量进行分析,在未感染组中,diptericin的表达量显著性降低(P<0.0001);而大肠杆菌和白色念珠菌感染后,diptericin的表达量显著提高(P<0.05);但金黄色葡萄球菌感染后其表达在两组间无统计学意义(P>0.05)。对家蝇defensin基因的表达量进行分析,在未感染组中,defensin的表达量显著性降低(P<0.05);而白色念珠菌、大肠杆菌及金黄色葡萄球菌感染后其表达在两组间均无统计学意义(P>0.05)。家蝇GNBP3基因在24 h时得到有效沉默并使抗菌肽基因的表达降低,细菌感染能够增高抗菌肽基因(cecropins、diptericin)的表达,而真菌感染时抗菌肽基因的表达各不相同。推测各抗菌肽基因间呈协同调节抗真菌的作用,且家蝇GNBP3基因在抗真菌免疫中起到一定作用,但可能同时存在其它抗真菌途径以共同完成家蝇先天性免疫系统对真菌的免疫应答。

To study the functional roles of housefly larvae GNBP3 in response to microorganism infection and evaluate the effects on downstream AMP levels,we used RNA interference to inhibit the production of GNBP3 and measured the interference effect with quantitative real-time PCR.Then we evaluated the challenge with exogenous microbes.Through GNBP3 dsRNA microinjection,GNBP3 was knocked down in the first 24 h after the injection,the survival rate and pupation rate of GNBP3-depleted housefly larvae was severely suppressed compared with GFP dsRNA and blank control group(P<0.05).The expression of AMP genes(cecropins,defensin,diptericin)was lower than those of control group after GNBP3 RNA interference(P<0.05).But its expression varied after exogenous microbes infection,its low expression with cecropins gene and high expression with diptericin gene after fungal infections compared with those of control,high expression(cecropins,diptericin)with grampositive and negative bacteria infections(P<0.05).GNBP3 gene of housefiy larvae was effectively silenced at 24 h after RNAi,that could cause a low expression of antibacterial peptide genes in the blank control.The highly expression(cecropins,diptericin)with bacteria infections and difference expression with fungal infection.It was speculated that among various antibacterial peptide gene was synergy to adjust the action of antifungal,and housefly GNBP3 gene played a significance role in the antifungal immunity,but there also were other antifungal ways at the same time for adjustments of housefly innate immune system to identification of fungi and immune response.