Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase.
Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase.
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DOI：
10.1186/1756-3305-3-73
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发表时间：
2010-08-16
影响因子：
3.2
通讯作者：
Bowman AS
中科院分区：
文献类型：
--
作者：
Campbell EM;Budge GE;Bowman AS
寄生性螨虫被认为是欧洲蜜蜂（Apis mellifera）的主要害虫，是全球蜜蜂数量下降的原因。利用基因序列的全部潜力成为可用于V. destructor需要现代分子生物学方法适应这种非模式生物。以一个mu类谷胱甘肽s -转移酶（VdGST-mu1）为候选基因，研究了双链rna干扰法（dsRNAi）敲低V. destructor基因的可行性。与注射大量不相关的dsRNA （LacZ）相比，腹腔内注射dsRNA-VdGST-mu1导致注射后48小时VdGST-mu1转录物水平降低97%。这种基因抑制至少维持了72小时。VdGST-mu1基因敲除螨的总GST催化活性降低了54%，表明敲除在翻译步骤和转录步骤都有效。虽然通过腹腔内注射实现了几乎完全的基因敲除，但只有一半的处理过的螨虫在这种创伤性的dsRNA给药方法中存活下来，并且评估了侵入性较小的方法。在含有dsRNA的0.9% NaCl溶液中浸泡过夜的破坏性螨显示出VdGST-mu1转录物水平的显著降低（与浸泡在dsRNA- lacz中的螨相比，降低了87%）。重要的是，经过浸泡处理的螨虫在72小时内的存活率（75-80%）大大提高，接近未经过任何处理的螨虫存活率。我们的研究结果是首次报道在任何螨虫物种中基因敲低，并表明这种生物的小尺寸并不是将基因敲低方法应用于此类寄生害虫研究的主要障碍。dsRNA溶液浸泡法是一种简便、廉价、相对高通量的基因沉默方法，适用于灭螨、其他小螨和蜱未成熟阶段的研究。
The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1) as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi). Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ). This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ). Importantly, mites undergoing the immersion approach had greatly improved survival (75-80%) over 72 h, approaching that of mites not undergoing any treatment. Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and immature stages of ticks.
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影响因子：
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