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

Abstract

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

CLC Number:

S718.7

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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Effects of Neuropeptide F and Its Receptor on Feeding, Growth and Reproductive of Hyphantria cunea (Lepidoptera: Arctiidae) with RNAi Technique

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