Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge.
Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge.
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DOI：
10.1371/journal.pone.0135093
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Franta Z
中科院分区：
文献类型：
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作者：
Baumann A;Lehmann R;Beckert A;Vilcinskas A;Franta Z
常见的绿瓶蝇（双翅目：绢绿蝇科）的幼虫已被用于促进伤口愈合几个世纪，但其抗菌、清创和愈合功能的分子基础仍不清楚。免疫攻击前后特异性幼虫组织中差异基因表达的分析可用于鉴定关键分子因子，但最敏感和可重复性最高的方法qRT-PCR需要经过验证的内参基因。因此，我们选择了10个候选内参基因，编码不同功能类别的产物（18S rRNA、28S rRNA、肌动蛋白、β-微管蛋白、RPS3、RPLP0、EF1α、PKA、GAPDH和GST1）。使用两种广泛应用的算法（GeNorm和Normfinder）分析与分泌、消化和抗菌活性相关的不同幼虫组织（中肠、后肠、唾液腺、作物和脂肪体）中的内参候选基因。然后使用革兰氏阴性细菌铜绿假单胞菌来增强幼虫的免疫系统，并与针对不同病原体类别的三种免疫基因（lucimycin，防御素-1和攻击素-2）进行比较，测试参考基因表达的稳定性。我们观察到抗真菌肽lucimycin的表达无差异，而针对革兰氏阳性细菌的代表性靶向菌（defenin -1）在唾液腺、嗉囊、神经节中表达上调，在脂肪体中达到最大值（高达300倍）。在所有免疫挑战组织中，最强烈的上调（在脂肪体中诱导超过50,000倍）被监测到针对革兰氏阴性菌的代表性攻击蛋白-2。在这里，我们鉴定并验证了一组内参基因，这些内参基因可以在免疫攻击后使丝光l.s icata特定组织中的基因表达准确正常化。
The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge.