RNA干扰分析神经肽F基因及其受体对美国白蛾取食、生长发育和生殖的影响

doi:10.11707/j.1001-7488.20221206

摘要/Abstract

摘要：

目的: 通过对美国白蛾及其受体(NPFR)基因进行克隆及功能分析，探究NPF和NPFR基因对美国白蛾取食、生长发育以及产卵的影响，旨在为开发靶向NPF和NPFR基因的害虫防治提供理论依据。方法: 克隆获得美国白蛾NPF和NPFR基因cDNA全长，采用RT-qPCR检测NPF/NPFR基因在不同发育阶段及各组织中的表达量，利用RNAi技术探讨NPF/NPFR基因对美国白蛾生长发育和生殖以及饥饿耐受性的调控作用。结果: 美国白蛾NPF和NPFR基因开放阅读框(ORF)分别为246 bp和1 176 bp，编码81个和391个氨基酸。不同发育阶段的表达模式显示HcNPF和HcNPFR基因分别在4龄幼虫和雄成虫表达量最高，分别为对照的79.62倍和2.96倍；组织特异性表达分析表明HcNPF和HcNPFR基因在中肠的表达量最高，分别为头部的3.18倍和27.09倍。与注射dsRed对照组相比，注射HcNPF和HcNPFR dsRNA在幼虫期和蛹期均有效沉默，且在96 h幼虫期沉默效率分别为32.71%和88.64%，蛹期沉默效率分别为85.68%和93.01%。幼虫期注射dsNFP和dsNPFR后取食量明显降低，dsNFP注射96 h较dsRed对照组降低了12.77%；dsNPFR处理144 h较dsRed对照组降低了21.83%，且体质量累计增长率在注射192 h后分别降低了30.89%和13.54%；靶基因沉默组(dsNFP和dsNPFR)成虫交配后平均产卵量显著低于dsRed对照组，依次为dsRed对照组的29.09%和40.22%；饥饿胁迫下注射NPF和NPFR dsRNA的美国白蛾幼虫死亡率显著增加，分别在96 h和102 h全部死亡，而对照组dsRed在96 h和102 h的死亡率分别为55.00%和58.33%。结论: 获得HcNPF和HcNPFR基因全长；在不同发育阶段与组织中HcNPF和HcNPFR表达具有特异性；沉默HcNPF和HcNPFR基因导致美国白蛾取食量和体质量累计增长率降低，对饥饿胁迫的敏感性增强。综上所述，NPF和NPFR基因在美国白蛾取食、生长发育和生殖功能方面发挥重要的作用，NPF和NPFR可作为美国白蛾绿色防治的分子靶标。

关键词: 美国白蛾, 神经肽F, G蛋白偶联受体, RNA干扰, 生理功能

Abstract:

Objective: Insect neuropeptides F (NPF) regulates physiological functions, such as feeding, development and reproduction, of insects by binding to its specific receptors. In this study, the effects of NPF and the NPF receptor (NPFR) genes on feeding, growth and reproduction of Hyphantria cunea were explored by cloning and functional analysis of NPF and NPFR, which would provide a theoretical foundation for further pest control targeting NPF and NPFR genes. Method: The full-length cDNA of NPF and NPFR were cloned from H. cunea. The expression levels of NPF/NPFR in different developmental stages and tissues were determined by RT-qPCR. The RNAi technique was used to analyze the regulatory effects of NPF/NPFR on feeding, growth development, reproduction and hunger tolerance. Result: The open reading frame (ORF) of NPF and NPFR was 246 bp and 1 176 bp, encoding 81 and 391 amino acids, respectively. The expression patterns at different developmental stages showed that NPF and NPFR had the highest expression levels in the 4^th instar larvae and male adults, and they were 79.62- and 2.96-fold higher than those in the control, respectively. Tissue specific expression analysis showed that the expression of HcNPF and HcNPFR were the highest in midgut, which were 3.18- and 27.09-fold higher than that in head, respectively. Compared control group injected with dsRed, dsRNA in the treatment group injected with HcNPF and HcNPFR was effectively silenced, and the silencing efficiency was 32.71% and 88.64% at 96 h larval stage, and 85.68% and 93.01% at pupal stage, respectively. The NFP and NPFR gene silencing reduced the food intake of larvae. The feeding of larvae treated by dsNFP was 12.77% lower than that of dsRed control group at 96 h, and the feeding of larvae treated by dsNPFR was 21.83% lower than that of dsRed control group at 144 h. The accumulative rate of body weight was 30.89% and 13.54% lower than that of the control at 192 h, respectively. The average egg production of adults in the silencing group (dsNFP and dsNPFR) after mating was significantly lower than that of the dsRed control group, which was 29.09% and 40.22% of that in the dsRed control group, respectively. Under starvation stress, the mortality of dsNPF and dsNPFR treatment were significantly higher than that of the control group, and all larvae died at 96 h and 102 h, while the mortality of dsRed were 55.00% and 58.33% at 96 h and 102 h, respectively. Conclusion: The full length of HcNPF and HcNPFR has been obtained, and the expression of HcNPF and HcNPFR is specific in different developmental stages and tissues. The feeding amount and acumulative growth rate of body weight are decreased by silencing HcNPF and HcNPFR, and the silenced larvae are more sensitive to starvation stress. In summary, HcNPF and HcNPFR play important roles in feeding, growth and reproductive of H. cunea, NPF and NPFR can be used as molecular targets for friendly control of H. cunea.

Key words: Hyphantria cunea, neuropeptide F, G protein-coupled receptors, RNA interference, physiological function

中图分类号:

S718.7

王晓琪,孙丽丽,殷晶晶,曹传旺. RNA干扰分析神经肽F基因及其受体对美国白蛾取食、生长发育和生殖的影响[J]. 林业科学, 2022, 58(12): 52-61.

Xiaoqi Wang,Lili Sun,Jingjing Yin,Chuanwang Cao. Effects of Neuropeptide F and Its Receptor on Feeding, Growth and Reproductive of Hyphantria cunea (Lepidoptera: Arctiidae) with RNAi Technique[J]. Scientia Silvae Sinicae, 2022, 58(12): 52-61.

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基因 Gene	正向引物序列(5’-3’) Forward primer sequences(5’-3’)	反向引物序列(5’-3’) Reverse primer sequences(5’-3’)	引物用途 Primer usage
HcNPF	ATGTTGAACAAGAACATTGCTATTGCTTTTGCCG	TCATCTCCTTCTCGCGTAAGACAGCC	扩增目的基因 Amplified target gene
HcNPFR	ATGCCTTTTTATGATGAAATGCCCCAC	GAAAATTTGAGAACACTGTGATGTC	扩增目的基因 Amplified target gene
HcNPF	ATGTTGAACAAGAACATTGCTATTGCTTTTGCCG	TCATCTCCTTCTCGCGTAAGACAGCC	实时荧光定量PCR RT-qPCR
HcNPFR	CGTCTATCCAACGCGACCA	ACATAGCTCGCCCATTCTCG
EF1α	ATGAAATCTCTGTGACCGGGG	GCGGTGGTATCGACAAACGT
RPL13	GTTAGCTACACAGCTCCGTGG	GCAGCAGTTGGGGCTTTAGT
dsHcNPF	TAATACGACTCACTATAGGG ATGTTGAACAAGAACATTGCTATTGCTTTTGCCG	TAATACGACTCACTATAGGG TCATCTCCTTCTCGCGTAAGACAGCC	合成dsRNA Synthesis of dsRNA
dsHcNPFR	TAATACGACTCACTATAGGG ATGCCCCACAACAACATGGA	TAATACGACTCACTATAGGG CAAATATAGTGGAGATGCAAGGGT
dsRed	TAATACGACTCACTATAGGG GAGAACGTCATCACCGAGTT	TAATACGACTCACTATAGGG GATGGTGTAGTCCTCGTTGT

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