经白色念珠菌胁迫后家蝇新型抗菌肽AMP17表达模式的研究

Expression pattern of a novel antibacterial peptide AMP17 from Musca domestica after Candida albicans stress

目的:探讨家蝇幼虫经白色念珠菌Candida albican胁迫后,AMP17基因时空表达谱的变化。方法:显微注射法将白色念珠菌导入家蝇幼虫体内,饲养3 h、6 h、12 h、24 h、36 h、48 h后,提取各组整虫RNA,采用实时荧光定量PCR分析AMP17的时间表达情况;选取经胁迫后3 h、12 h、48 h的幼虫,解剖获得体壁、脂肪体、马氏管、中肠、唾液腺、气管、血淋巴,同上分析AMP17的空间表达情况。以注射PBS为对照组,家蝇RPS18为内参基因,设3个生物重复。结果:注射白色念珠菌后,实验组幼虫的AMP17基因表达量较对照组明显上调,高表达一直持续到12 h,24 h明显下降,但48 h表达量又突然大幅度升高。不同组织中实验组与对照组比较,3 h时体壁表达量最高,上调6. 59倍(P<0. 01),其次是气管上调4. 74倍;感染12 h后,体壁表达量依然最高,上调了5. 54倍(P<0. 01),其次为血淋巴,上调了1. 87倍(P<0. 05);感染48 h后,体壁表达量上调了10. 98倍(P<0. 05);唾液腺上调了7. 28倍(P<0. 05)。结论:经白色念珠菌胁迫后,AMP17基因的表达量出现了明显的上调,提示该基因参与了家蝇应对真菌侵染的免疫反应。

Objective:To investigate the spatial and temporal expression profile of AMP17 gene in Musca domestica larvae after stress by Candida albican.Methods:Candida albicans infects Musca domestica larvae.After feeding for 3 h,6 h,12 h,24 h,36 h,and 48 h,RNA of each group was extracted and real-time fluorescence quantitative PCR was used to analyze the temporal expression of AMP17.The larvae at 3 h,12 h and 48 h after stress were selected to obtain body wall,fat body,malpighian tubule,midgut,salivary gland,trachea and hemolymph.The spatial expression of AMP17 was analyzed as above.Injected PBS as control group,Musca domestica RPS18 as an internal reference gene with 3 biological replicates.Results:After injection of Candida albicans,the expression level of AMP17 gene in experimental group larvae was significantly higher than that in the control group.The high expression lasted until 12 h and decreased significantly at 24 h,but the expression level suddenly increased sharply at 48 h.Compared with the control group,the expression of body wall was the highest at the 3 h,which was 6.59 times higher(P<0.01),followed by 4.74 times the trachea uptake;after 12 h,the body wall expression was still the highest,up 5.54 times(P<0.01),followed by hemolymph,increased 1.87-fold(P<0.05);after 48 h of infection,body wall expression was up-regulated by 10.98-fold(P<0.05);salivary glands were up-regulated by 7.28-fold(P<0.05).Conclusion:After being challenged by Candida albicans,the expression of AMP17 gene was significantly up-regulated,suggesting that this gene is involved in the immune response of Musca domestica against fungal infection.