热带病与寄生虫学

热带病与寄生虫学 ›› 2024, Vol. 22 ›› Issue (6): 346-352.doi: 10.20199/j.issn.1672-2302.2024.06.005

• 病媒生物防制专题 • 上一篇    下一篇

RNA干扰乙酰胆碱酯酶基因对福寿螺生长及存活的影响

张健1,2(), 开振鹏2, 程东慧1, 郭云海1(), 张仪1,3()   

  1. 1 传染病溯源预警与智能决策全国重点实验室，中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心），国家卫生健康委员会寄生虫病原与媒介生物学重点实验室，世界卫生组织热带病合作中心，科技部国家级热带病国际研究中心，上海 200025
    2 上海应用技术大学化学与环境工程学院
    3 上海交通大学医学院-国家热带病研究中心全球健康学院
  • 收稿日期:2024-09-26 出版日期:2024-12-20 发布日期:2025-01-23
  • 通信作者: 郭云海,guoyh@nipd.chinacdc.cn;张仪,zhangyi@nipd.chinacdc.cn
  • 作者简介:张健，女，硕士在读，研究方向：生态学与病媒控制研究。E-mail: 862112019@qq.com
  • 基金资助:
    国家重点研发计划项目(2021YFC2300800);国家重点研发计划项目(2021YFC2300802);国家自然科学基金合作项目(82161160343)

Impact of RNA interference of acetylcholinesterase gene on the growth and survival of Pomacea canaliculata

ZHANG Jian1,2(), KAI Zhenpeng2, CHENG Donghui1, GUO Yunhai1(), ZHANG Yi1,3()   

  1. 1 National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese National Tropical Diseases Research Center); Key Laboratory on Parasite and Vector Biology, Ministry of Health; WHO Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China
    2 School of Chemical and Environmental Engineering, Shanghai Institute of Technology
    3 School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine
  • Received:2024-09-26 Online:2024-12-20 Published:2025-01-23
  • Contact: GUO Yunhai,guoyh@nipd.chinacdc.cn;ZHANG Yi,zhangyi@nipd.chinacdc.cn

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摘要：

目的 观察利用RNA干扰（RNA interference, RNAi）技术干扰福寿螺乙酰胆碱酯酶（acetylcholinesterase, AChE）基因表达对福寿螺生长和生存的影响，探究RNAi技术在福寿螺防制中的应用价值。方法 针对小管福寿螺AChE基因设计双链RNA（double-stranded RNA, dsRNA），构建相应质粒并分别转化到大肠杆菌工程菌中。研究分为空白对照组、阴性对照组和实验组，其中实验组又分为F1、F2、F3、F4组，每组10只福寿螺，空白对照组福寿螺不予处理，阴性对照组给福寿螺喂食空载大肠杆菌，F1、F2、F3、F4组给福寿螺分别喂食4种不同AChE-dsRNA诱导后的工程菌，利用实时荧光定量PCR（RT-qPCR）检测8、12、24 h福寿螺AChE表达水平。选择使福寿螺AChE表达水平变化最大的工程菌，对福寿螺进行喂食和注射，观察福寿螺生长和存活情况。结果 给福寿螺喂食表达AChE-dsRNA的工程菌8 h和12 h后，与阴性对照组相比，F1、F3、F4组AChE表达水平未见明显影响，F2组AChE表达水平下降（t=5.790、10.110，P均<0.01）;喂食24 h后， 各实验组AChE的表达均未受影响。连续喂食表达AChE-dsRNA-F2片段的工程菌30 d后，福寿螺未发生死亡或明显的表型变化，平均体重由（0.560 9 ± 0.113 6）g增长到（0.655 9 ± 0.177 3）g，体重增长16.9%;而喂食空载工程菌的阴性对照组平均体重由（0.565 0 ± 0.131 7）g增长到（0.691 2 ± 0.156 8）g，体重增长23.3%;两组体重增加差异有统计学意义（F=2.720，P<0.05）。注射表达AChE-dsRNA-F2干扰片段的工程菌后，福寿螺死亡率为30.0%，而注射空载工程菌的福寿螺死亡率为13.3%，差异有统计学意义（χ2=4.910，P<0.05）。结论 喂食 AChE-dsRNA诱导的工程菌能有效降低福寿螺AChE基因的表达水平并抑制其体重增长，而注射 AChE-dsRNA诱导的工程菌可增加福寿螺的死亡率，RNAi技术为福寿螺的防制提供了一种潜在思路。

关键词: 福寿螺, RNA干扰, 乙酰胆碱酯酶基因, 双链RNA

Abstract:

Objective To investigate the impact of RNA interference (RNAi) on the expression of the acetylcholinesterase (AChE) gene in Pomacea canaliculata and its effects on the growth and survival of the snail for potential application of RNAi technology in the control of this pest. Methods Double-stranded RNA (dsRNA) targeting the AChE gene of P. canaliculata was designed, and the corresponding plasmid was constructed and transformed into Escherichia coli to create engineered bacteria. The study included a blank control group, a negative control group, and experimental groups (F1, F2, F3, F4), with 10 snails in each group. The blank control group received no treatment, the negative control group was fed with E. coli carrying an empty vector, and the experimental groups F1, F2, F3, and F4 were fed with the engineered E. coli to express different AChE-dsRNAs. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of AChE at 8, 12, and 24 hours post-treatment. The group showing the most significant change in AChE expression was selected for further feeding and injection tests to observe the survival and mortality of the snails. Results After feeding the snails with E. coli expressing AChE-dsRNA for 8 and 12 hours, group F2 showed a significant decrease in AChE expression (t=5.790, 10.110, P<0.01) compared to the negative control group, whereas groups F1, F3, and F4 did not show significant changes. After 24 hours of feeding, AChE expression was unaffected in the snails in each experimental group. Further feeding with E. coli expressing AChE-dsRNA-F2 for 30 days, the average weight of the snails in group F2 was increased by 16.9% [ranging from (0.560 9 ± 0.113 6) g to (0.655 9 ± 0.177 3) g], and the average weight of the snails in the negative control group was increased by 23.3%[ranging from (0.565 0 ± 0.131 7) g to (0.691 2 ± 0.156 8) g]. The difference in weight gain was statistically significant in both groups (F=2.720, P<0.05). Injection with E. coli expressing AChE-dsRNA-F2 led to a 30.0% mortality of the snails, compared to 13.3% in the control group, with significant difference between groups (χ2=4.910, P<0.05). Conclusion Feeding E. coli engineered to express AChE-dsRNA can effectively reduce the expression of the AChE gene in P. canaliculata and inhibit its weight gain, and injection with AChE-dsRNA-expressing E. coli may increase the mortality. Our findings suggest that RNAi technology may be a novel rationale in the control of P. canaliculata.

Key words: Pomacea canaliculata, RNA interference (RNAi), Acetylcholinesterase gene, Double-stranded RNA

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