CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae)
CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae)
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CRISPR/Cas9 介导的 NlCSAD 基因敲除导致褐飞虱（半翅目：飞虱科）角质层色素沉着加深和雌性繁殖力下降
DOI：
10.1016/j.cbpa.2021.110921
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发表时间：
2021-03-02
影响因子：
2.3
通讯作者：
Zhang, Wen-Qing
中科院分区：
文献类型：
--
作者：
Chen, Jing-Xiang;Li, Wan-Xue;Zhang, Wen-Qing
在昆虫中，表皮色素基因已被开发为潜在的可见标记，用于构建遗传操作系统。在这里，我们克隆了半胱氨酸亚磺酸脱羧酶（CSAD），黑色素代谢途径基因的直向同源物，并在褐飞虱（半翅目：Delphacidae）中进行RNAi实验。结果显示，与对照组相比，NICSAD转录水平的降低增加了体内的黑色素沉积，导致更深的角质层色素沉着。用dsNlCSAD处理并与野生型雄性交配的雌性成虫比dsGFP处理组产下显著更少的卵，并且还观察到卵的孵化率更低。此外，还发现了两个黑素突变体N.通过CRISPR/Cas9基因组编辑系统构建的褐飞虱品系（NlCSAD（-/+）和NlCSAD（-/-））表现出较深的表皮黑化以及降低的产卵和孵化率，但纯合子在RNAi后具有比杂合子或个体更深的体色、更少的卵和更低的孵化率。因此，我们提供了第一个证据表明，NlCSAD是正常的身体色素沉着所必需的成年人，并在女性的生殖力和鸡蛋的孵化率在N。基于CRISPR/Cas9基因组编辑系统，通过RNAi和靶基因敲除的组合来改造褐飞虱。我们的研究结果表明，NlCSAD是一个候选的视觉参考基因的遗传操作这一重要的作物害虫。
In insects, cuticular pigmentation genes have been exploited as potential visible markers for constructing genetic manipulation systems. Here, we cloned cysteine sulfinic acid decarboxylase (CSAD), an orthologue of melanin metabolism pathway genes, and performed RNAi experiments in the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae). The results showed that a decrease in the level of transcription of NlCSAD increased melanin deposition in the body compared to the control group, resulting in darker cuticle pigmentation. Female adults treated with dsNlCSAD and mated with wild-type males laid significantly fewer eggs than the dsGFP-treated group, and lower hatchability of the eggs was also observed. In addition, two melanic mutant N. lugens strains (NlCSAD(-/+) and NlCSAD(-/-)) constructed by the CRISPR/Cas9 genome editing system showed darker cuticular melanisation and a reduced oviposition and hatching rate, but the homozygotes had a darker body colour, fewer eggs and lower hatchability than heterozygotes or individuals after RNAi. Thus, we have provided the first evidence that NlCSAD is required for normal body pigmentation in adults and has a role in the fecundity of females and hatchability of eggs in N. lugens via a combination of RNAi and knockout of target genes based on the CRISPR/Cas9 genome editing system. Our results suggest that NlCSAD is a candidate visual reference gene for genetic manipulation of this important crop pest.