CRISPR/Cas9介导的serpin15基因敲除对小菜蛾繁殖和免疫的影响

doi:10.1016/j.jia.2025.09.004

Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (8): 3330-3340.DOI: 10.1016/j.jia.2025.09.004

CRISPR/Cas9介导的serpin15基因敲除对小菜蛾繁殖和免疫的影响

收稿日期:2025-05-26 修回日期:2025-09-04 接受日期:2025-08-08 出版日期:2026-08-20 发布日期:2026-07-08

CRISPR/Cas9-mediated knockout of serpin15 impacts reproduction and immunity in Plutella xylostella Linnaeus

Yanbo Jia, Hongxin Wu, Yuting Huang, Yifan Liu, Shaojie Zhu, Zhantao Zhang, Junlin Huang, Junaid Zafar, Rui Pang, Xiaoxia Xu^#, Fengliang Jin^#

State Key Laboratory of Green Pesticide/“Belt and Road” Technology Industry and Innovation Institute for Green and Biological Control of Agricultural Pests/College of Plant Protection, South China Agricultural University, Guangzhou 510642, China

Received:2025-05-26 Revised:2025-09-04 Accepted:2025-08-08 Online:2026-08-20 Published:2026-07-08
About author:Yanbo Jia, Mobile: +86-17332542406; E-mail: jyb0202@stu.scau.edu.cn; Correspondence Xiaoxia Xu, Mobile: +86-13560177128; E-mail: xuxiaoxia111@scau.edu.cn; Fengliang Jin, Mobile: +86-13560478369; E-mail: jflbang@scau.edu.cn
Supported by:
This work was supported by grants from the National Natural Science Foundation of China (32172498, 31972345 and W2433052), and the Natural Science Foundation of Guangdong, China (2023A1515010305).

摘要/Abstract

摘要：

小菜蛾（Plutella xylostella）是一种危害全球十字花科作物的毁灭性害虫。对化学合成杀虫剂的过度依赖导致了生态污染和抗药性的产生，这促使研究者探索环境友好型生物农药。丝氨酸蛋白酶抑制剂（serpins）在先天免疫中的黑化作用、繁殖以及变态发育中发挥着关键作用。基于我们实验室对小菜蛾不同发育阶段进行的蛋白质组学分析，serpin15被确定为典型抑制性serpin家族的关键成员，但其在小菜蛾中的具体功能仍不明确。在本研究中，通过对不同组织和发育阶段的基因表达模式进行RT-qPCR分析发现，serpin15在雄性成虫的性腺中高表达，且在血淋巴中的丰度最高。值得注意的是，在被粘质沙雷氏菌（PS-1）感染后，中肠内serpin15的mRNA水平呈现动态变化，先下降后上调。利用CRISPR/Cas9技术在纯合品系中敲除serpin15后，产卵率和后代的胚胎孵化率均有所降低。功能实验证实，serpin15能抑制酚氧化酶（PO）的活性，外源性补充重组serpin15蛋白可有效抑制血淋巴的黑化，从而确立了其通过免疫黑化作用对抗PS-1的调控功能。综上所述，这些研究结果强调了serpin15在小菜蛾中具有调节繁殖力和对抗PS-1免疫力的双重功能。这一发现为开发针对昆虫免疫和发育系统的生物防治策略提供了理论基础。

Abstract:

Plutella xylostella represents a significant agricultural pest affecting cruciferous crops globally. The extensive use of synthetic insecticides has resulted in environmental contamination and resistance development, necessitating research into environmentally sustainable biopesticides. Serine protease inhibitors (serpins) serve essential functions in melanization during innate immunity, reproduction, and metamorphic development. Through proteomic analyses conducted across developmental stages of P. xylostella, serpin15 was identified as a crucial member of the typical inhibited serpin family, though its precise function remained undetermined. RT-qPCR analyses of gene expression patterns across tissues and developmental stages demonstrated that the serpin15 gene exhibits high expression in male adult gonads and reaches maximum levels in hemolymph. The serpin15 mRNA levels showed dynamic regulation in the midgut following Serratia marcescens (PS-1) infection, characterized by an initial decline followed by upregulation. CRISPR/Cas9-mediated knockout of serpin15 in homozygous lines led to decreased oviposition and embryonic hatching rates in offspring. Functional analyses confirmed that serpin15 inhibits phenoloxidase activity, while exogenous supplementation with recombinant serpin15 protein effectively suppressed hemolymph melanization, establishing its regulatory role in countering PS-1 through immune melanization. These findings demonstrate serpin15’s dual functionality in regulating both fecundity and immunity against PS-1 in P. xylostella. This research establishes a theoretical foundation for developing biocontrol strategies targeting insect immune and developmental systems.

Key words: Plutella xylostella , serine protease inhibitors , CRISPR/Cas9 , melanization

Yanbo Jia, Hongxin Wu, Yuting Huang, Yifan Liu, Shaojie Zhu, Zhantao Zhang, Junlin Huang, Junaid Zafar, Rui Pang, Xiaoxia Xu, Fengliang Jin. CRISPR/Cas9介导的serpin15基因敲除对小菜蛾繁殖和免疫的影响[J]. Journal of Integrative Agriculture, 2026, 25(8): 3330-3340.

Yanbo Jia, Hongxin Wu, Yuting Huang, Yifan Liu, Shaojie Zhu, Zhantao Zhang, Junlin Huang, Junaid Zafar, Rui Pang, Xiaoxia Xu, Fengliang Jin. CRISPR/Cas9-mediated knockout of serpin15 impacts reproduction and immunity in Plutella xylostella Linnaeus[J]. Journal of Integrative Agriculture, 2026, 25(8): 3330-3340.

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CRISPR/Cas9介导的serpin15基因敲除对小菜蛾繁殖和免疫的影响

CRISPR/Cas9-mediated knockout of serpin15 impacts reproduction and immunity in Plutella xylostella Linnaeus

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