Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Dept. of Developmental Biology, Georg-August-University Goettingen, GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Goettingen, 37077, Germany.
Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Dept. of Evolutionary Developmental Genetics, Georg-August-University Goettingen, GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Goettingen, 37077, Germany.
BMC Genomics. 2022 Aug 20;23(1):608. doi: 10.1186/s12864-022-08822-z.
Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before.
Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause phenotypic changes in the glands upon knock-down, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. Gene ontology analysis revealed that many of those genes are encoding enzymes (peptidases and cytochromes P450) as well as proteins involved in membrane trafficking with an enrichment in lysosome and mineral absorption pathways. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones in the gland reservoirs, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. However, only one out of eight genes identified by a transcriptomics approach known to cause phenotypic changes of the glands upon knock-down was recognized by this phenotypic screen, indicating the limitation of such a non-redundant first-pass screen.
Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.
功能基因组学使用无偏系统的全基因组基因敲除或分析多细胞生物不同组织的基因表达谱等自然变异，将基因功能与特定表型联系起来。功能基因组学方法对于鉴定大量特定于特定生物学过程的基因特别重要，这些基因超出了已知候选基因的范围，或者在以前的遗传方法研究之前该过程尚未被研究过。
在这里，我们展示了一组大量的基因，这些基因的破坏会干扰赤拟谷盗（Tribolium castaneum）有气味的防御性臭腺的功能。这个基因集是使用 RNA 干扰进行全基因组正向遗传学大规模系统表型筛选的结果。在这个首次筛选中，鉴定了 130 个基因，其中 69 个基因在敲低时可以证实导致腺体表型发生变化，这些变化从坏死组织和不规则储库大小到不规则颜色或分泌腺化合物的分离不等。基因本体论分析表明，其中许多基因编码酶（肽酶和细胞色素 P450）以及参与膜运输的蛋白质，并且富含溶酶体和矿物质吸收途径。13 个基因的敲低导致腺体储库中的对苯醌明显减少，表明这些有毒化合物的合成具有特定的功能。在腺体组织中，69 个经证实的腺体基因中只有 14 个差异过表达，因此可以在基于转录组的分析中检测到。然而，在通过敲低导致腺体表型发生变化的 8 个已知基因中，只有一个通过转录组学方法鉴定的基因被这种表型筛选所识别，这表明这种非冗余首次筛选的局限性。
我们的结果表明，结合不同的和独立的方法来鉴定对于特定生物组织的功能所必需的基因非常重要，因为不同的方法不会提供冗余的结果，而是相互补充。本文所呈现的表型筛选与转录组学方法一起，为甲虫有气味的防御性臭腺生理学提供了近百个重要基因。
