CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura
CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura
复制标题
CRISPR/Cas9介导的基因敲除揭示了PBP1在斜纹夜蛾雌性性信息素成分感知中比PBP2更重要的作用
DOI：
10.1016/j.ibmb.2019.103244
复制
发表时间：
2019-12-01
影响因子：
3.8
通讯作者：
Dong, Shuang-Lin
中科院分区：
文献类型：
--
作者：
Zhu, Guan-Heng;Zheng, Mei-Yan;Dong, Shuang-Lin
通常可以在夜间蛾触角的感觉淋巴中找到三种不同的信息素结合蛋白（PBP），但是在体内很少证实它们在性信息素的感知中的相对贡献。以前，我们证明了使用CRISPR/CAS9系统在Spodoptera Litura的性信息素检测中扮演较小的角色。在本研究中，使用同一系统进一步验证了另外两个SlitPBP（Slitpbp1和Slitpbp2）的作用。首先，通过将Cas9 mRNA/sgrna共入卵中，诱导了高靶诱变率，SLITPBP2的SAPBP1为51.5％，由限制性酶测定确定。然后，通过交叉繁殖获得了纯合slitpbp1和slitpbp2敲除线。最后，使用纯合敲除雄性飞蛾，我们进行了电生理（EAG记录）和行为分析。结果表明，男性中的SlitPBP1或SlitPBP2敲除对3个性别信息酮成分（Z9，E11-14：AC，Z9，E12-14：AC和Z9-14：AC）的3个性别信息组件的反应降低了53％，60％和63％（对于SLITPBB1敲除）和46％，43％（46％）（si）和46％（46％）（46％）（46％）（ 分别。 EAG反应中的这些降低在3个信息素组件中相似，但在SLITPBP1基因敲除男性中比SIITPBP2基因敲除男性更为明显。一致地，使用主要成分（Z9，E11-14：AC）的行为分析表明，SlitpBP1基因敲除男性的响应百分比要低于SLITPBP2基因敲除男性，而对于信息素的方向而言，以及近距离行为的降低，例如发hairpencil表现和近距离行为。综上所述，这项研究提供了直接的功能证据，证明了SLITPBP1和SIITPBP2的作用，以及它们相对重要性（SLITPBP1> SLITPBP2）在性信息素感知中。该信息对于理解性信息素感知的机制很有价值，并可能促进靶向PBP的害虫控制技术的发展。
Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SaPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SIitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SIitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SIitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.