橘小实蝇转基因系统的开发及其在嗅觉靶标研究中的应用潜力

doi:10.1016/j.jia.2024.07.021

摘要/Abstract

摘要：

害虫行为调控剂旨在通过影响害虫的行为来减少对农作物的伤害，其中参与嗅觉行为的功能基因是研发害虫行为调控剂的关键分子靶标。在橘小实蝇（Bactrocera dorsalis）中，当前此方面的基因编辑研究主要依靠CRISPR/Cas9介导的敲除技术，这在致死基因的研究上受到了限制。转基因技术能够在特定组织或时期精确操控基因，解决了此局限。因此，本研究通过piggyBac介导的随机插入技术，构建了橘小实蝇的转基因操作系统，并测试了报告基因在嗅觉器官表达的模式，以及其对嗅觉行为和电生理表型的影响，以评估该系统在未来嗅觉基因功能研究中的应用潜力。首先，我们通过发育时期转录组鉴定到了在各时期广泛表达的管家基因BdorActinA3a-1，随后将此基因的候选启动子区域BdorActinA3a-1^P-2k克隆至piggyBac转基因质粒上，并以此构建获得两个稳定遗传的转基因品系，转基因片段分别插入到4号和5号染色体上。转基因品系的生存能力基本正常，插入元件不影响品系的孵化率和成虫生长曲线，但降低了羽化率和产卵量。在表达模式上，报告基因被有效在触角，口器，下颚须，足，外生殖器以及脑部的驱动表达。转基因品系对重要化合物的触角电位反应与野生型一致。但一些与嗅觉相关的行为受到了显著影响，例如对性信息素2,3,5-三甲基吡嗪（2,3,5-trimethylpyrazine,TMP）的反应和交配行为。这些结果说明该系统在未来可用于嗅觉基因功能的研究，例如在嗅觉组织中驱动一些嗅觉功能研究常用的元件表达。但应用时应注意它对一些嗅觉表型的影响。

Abstract:

Chemicals that modify pest behavior are developed to reduce crop damage by altering pest behavior, using specific genes within the olfactory system as molecular targets. The identification of these molecular targets in Bactrocera dorsalis, also known as the functional study of key olfactory genes, relies on CRISPR/Cas9-mediated gene knockout techniques. However, these techniques face limitations when applied to lethal genes. Transgenic technology offers a solution since it enables precise manipulation of gene expression in specific tissues or during certain developmental stages. Consequently, this study developed a piggyBac-mediated transgenic system in B. dorsalis to investigate reporter gene expression in olfactory organs, and assessed the olfactory behavior and antennal electrophysiological responses in transgenic lines. The goal was to assess the potential of this approach for future research on olfactory gene function. A universally expressed housekeeping gene from the BdorActin family was identified using the developmental transcriptome dataset. Its candidate promoter region (BdorActinA3a-1^P-2k) was then cloned into the piggyBac plasmid. We subsequently established two stable transgenic lines with specific TTAA insertion sites on chromosomes 4 and 5, consistent with the characteristics of piggyBac transposition. The transgenic strains exhibited essentially normal survival, with hatchability and adult lifespan unaffected, although there were slight reductions in the emergence rate and oviposition capacity. The fluorescent reporter has been successfully expressed in olfactory-related organs, such as the antennae, proboscis, maxillary palp, legs, external genitalia, and brain. The antennal electrophysiological responses to representative chemicals in the transgenic lines were consistent with those of the wild type. However, some olfactory-related behaviors, such as pheromone response and mating, were significantly affected in the transgenic lines. These findings suggest that our system could potentially be applied in future olfactory research, such as driving the expression of exogenous elements that are effective in olfactory organs. However, caution is advised regarding its impact when applied to some olfactory-related behavioral phenotypes.

Key words: olfactory , transgenic , piggyBac , Bactrocera dorsalis

. 橘小实蝇转基因系统的开发及其在嗅觉靶标研究中的应用潜力[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.07.021.

Jie Zhang, Qi Wang, Jinxi Yuan, Zhen Tian, Shanchun Yan, Wei Liu, Guirong Wang.

Development of a piggyBac transgenic system in Bactrocera dorsalis and its potential for research on olfactory molecular targets

[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.07.021.

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