Analysis of the Gene Expression and RNAi-Mediated Knockdown of Chitin Synthase from Leaf-Cutting Ant Atta sexdens

Chitin synthase (CHS) is the enzyme specifically associated with chitin synthesis, an important component of diverse organisms including insects. Two alternative spliced transcripts of the CHS gene (AsCHS-A1 and AsCHS-A2) were identified by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) during the development of the leaf-cutting ant Atta sexdens. Expression profiles of AsCHS-A transcripts increased from larva to pupae and decay in workers. Phylogenetic analysis showed both transcripts are classified within class A insect CHSs. AsCHS-A1 showed the highest expression level in larvae and pupae, while AsCHS-A2 is the main CHS transcript in workers. Our results suggest that these variants should be under regulation of different promoters. AsCHS-A1 has topology expected for insect CHSs, while the predicted AsCHS-A2 topology, with a missing A domain, is similar to some fungal CHSs. CHS-B (class B) was not identified in A. sexdens transcriptome. Ribonucleic acid interference (RNAi)-mediated gene silencing in pupae revealed that low reduction in CHS transcript levels (18%) was enough to cause morphological changes in the pupa exoskeleton impairing the process of cuticle sclerotization. To our knowledge, this work was the first to use and to show the feasibility of using RNA interference techniques on leaf-cutting ants.

几丁质合酶（Chitin synthase, CHS）是与几丁质合成特异性相关的酶，而几丁质是包括昆虫在内的多种生物的重要组成成分。本研究通过定量反转录聚合酶链式反应（quantitative reverse-transcription polymerase chain reaction, RT-qPCR），在切叶蚁Atta sexdens的发育过程中，鉴定得到了CHS基因的两种可变剪接转录本（AsCHS-A1与AsCHS-A2）。AsCHS-A转录本的表达水平从幼虫期至蛹期逐步升高，在工蚁体内出现表达下调。系统发育分析显示，两种转录本均归属于昆虫A型几丁质合酶。AsCHS-A1在幼虫与蛹期呈现最高表达水平，而AsCHS-A2则是工蚁体内的主要几丁质合酶转录本。本研究结果表明，这两种可变剪接变体可能受不同启动子调控。AsCHS-A1具备昆虫几丁质合酶预期的拓扑结构，而预测得到的AsCHS-A2拓扑结构缺失了A结构域，与部分真菌几丁质合酶的拓扑结构相似。在Atta sexdens的转录组中未鉴定到CHS-B（B型几丁质合酶）。通过核糖核酸干扰（ribonucleic acid interference, RNAi）介导的基因沉默技术处理蛹体，结果显示，仅需将几丁质合酶转录本水平降低18%，即可引发蛹体外骨骼的形态变化，阻碍表皮鞣化过程。据我们所知，本研究首次在切叶蚁中应用并验证了核糖核酸干扰技术的可行性。