GTP环化水解酶——一种防治褐飞虱(Nilaparvata lugens)的RNAi靶标基因

doi:10.3969/j.issn.1004-1524.20240530

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (6): 1285-1292.DOI: 10.3969/j.issn.1004-1524.20240530

GTP环化水解酶——一种防治褐飞虱(Nilaparvata lugens)的RNAi靶标基因

董代幸¹(), 羊桂英², 王爱英², 罗举², 刘淑华²^,^*()

1.杭州市富阳区农业技术推广中心,浙江杭州 311400
2.中国水稻研究所,浙江杭州 311401

收稿日期:2024-06-21 出版日期:2025-06-25 发布日期:2025-07-08
作者简介:董代幸(1984—),男,浙江温州人,硕士,农艺师,主要从事植物病虫害研究与技术推广。E-mail:15167120905@163.com
通讯作者: *刘淑华,E-mail:liushuhua@caas.cn
基金资助:
浙江省农业产业技术团队项目

GTP cyclohydrolase: a RNAi target gene against the brown planthopper (Nilaparvata lugens)

DONG Daixing¹(), YANG Guiying², WANG Aiying², LUO Ju², LIU Shuhua²^,^*()

1. Fuyang District Agricultural Technology Extension Center, Hangzhou City, Hangzhou 311400, China
2. China National Rice Research Institute, Hangzhou 311401, China

Received:2024-06-21 Online:2025-06-25 Published:2025-07-08

摘要/Abstract

摘要： 磷酸鸟苷环化水解酶I(guanosine triphosphate cyclohydrolase I, GCHI)是全变态昆虫重要的生长发育调节因子,参与黑色素形成和蜕皮过程。为探讨了GCHI编码基因作为褐飞虱RNA干扰(RNA interference, RNAi)防控靶标基因的可行性,克隆了褐飞虱的NlGCHI基因,分析了其时空表达特征,并采用显微注射方式进行了功能验证。结果表明,NlGCHI有2个转录本,分别命名为NlGCHIa和NlGCHIb。其中,NlGCHIb在高龄褐飞虱若虫期存在周期性表达动态,且在表皮中表达水平最高。无论是注射dsNlGCHI还是喂食GCHI抑制剂(3-deoxy-D-arabino-heptulosonate-7-phosphate, DAHP),褐飞虱都有极高的死亡率,主要是死于蜕皮障碍,表现为虫蜕不能顺利脱离虫体。dsNlGCHI处理试虫还表现出不能正常黑化的现象。以上结果表明,GCHI在全变态昆虫和不完全变态昆虫中的功能相对保守,影响昆虫的表皮形成和蜕皮过程,其编码基因可用于开发防治褐飞虱的RNAi农药。

关键词: 褐飞虱, RNA干扰, 磷酸鸟苷环化水解酶I, 表皮

Abstract:

Guanosine triphosphate cyclohydrolase I (GCHI) is a crucial regulatory factor for the growth and development of holometabolous insects, involved in melanin synthesis and the molting process. To evaluate the feasibility of the GCHI gene as a target for RNA interference (RNAi) in controlling the hemimetabolous insect Nilaparvata lugens(brown planthopper, BPH), this study cloned the NlGCHI gene from BPH, analyzed its spatiotemporal expression patterns, and validated gene function through microinjection experiments. The results indicated that NlGCHI has two transcripts, designated as NlGCHIa and NlGCHIb. Notably, NlGCHIb exhibited periodic expression dynamics in late-instar nymphs and was highly expressed in the cuticle. Treatment of BPH with dsNlGCHI injection or feeding with the GCHI inhibitor 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) resulted in high mortality rates, primarily due to molting defects characterized by failure to shed the old cuticle properly. Additionally, dsNlGCHI-treated individuals displayed abnormal melanization. The above results indicate that the function of GCHI remains relatively conserved in both holometabolous insects and hemimetabolous insect, influencing cuticle formation and molting processes in insects. Its encoding gene can be utilized to develop RNAi-based pesticides for controlling N. lugens.

Key words: Nilaparvata lugens, RNA interference, guanosine triphosphate cyclohydrolase I, cuticle

中图分类号:

S435.112.3

董代幸, 羊桂英, 王爱英, 罗举, 刘淑华. GTP环化水解酶——一种防治褐飞虱(Nilaparvata lugens)的RNAi靶标基因[J]. 浙江农业学报, 2025, 37(6): 1285-1292.

DONG Daixing, YANG Guiying, WANG Aiying, LUO Ju, LIU Shuhua. GTP cyclohydrolase: a RNAi target gene against the brown planthopper (Nilaparvata lugens)[J]. Acta Agriculturae Zhejiangensis, 2025, 37(6): 1285-1292.

图/表 5

表1 本文所用特异性引物序列

Table 1 The specific primers used in this research

用途 Purpose	引物名称^a Primer name^a	引物序列(5'→ 3')^b Primer sequence(5'→ 3')^b
ORF扩增	NlGCHI-F1	ATGGAGCTGGACCACAGACCAC
ORF clone	NlGCHI-R1	CAAGAATTCCTCGCGTGTCTTGG
非翻译区扩增RACE	NlGCHI-5'	GTGGTCTGTGGTCCAGCTCCATGTC
	NlGCHI-3'	GCAAGACGACCACATCTACGATGCTG
实时荧光定量PCR	NlGCHIa-F	CACCAGGTCACGAGAAA
qRT-PCR	NlGCHIb-F	CGAGAATGGTCACGAGAA
	NlGCHIa&b-R	AATACGGCGTCATTCAG
	qRPS15-F	CCGATCGTGTGGCGTTGAAGGG
	qRPS15-R	ATGGCCGACATTCTTCCAGGTCC
	qTUB-F	ACTCGTTCGGAGGAGGCACC
	qTUB-R	GTTCCAGGGTGGTGTGGGTGGT
dsRNA合成引物	dsNlGCHI-F	taatacgactcactatagggagGCATCACTTGGTCCCATTC
dsRNA synthesis	dsNlGCHI-R	taatacgactcactatagggagAATTCCTCGCGTGTCTTGG

图1 褐飞虱中NlGCHIa和NlGCHIb的时空表达模式 (A)NlGCHIa和NlGCHIb在不同发育阶段的相对表达水平。N4-4h代表4 h日龄的四龄若虫,FA-4h代表4 h日龄的雌成虫,其他样本的命名规则类似。(B)2种转录本在不同组织中的相对表达水平。MG代表中肠;FB代表脂肪体;CT代表表皮;OV代表卵巢;TE代表精巢。相对表达水平为平均值±标准误(n=4)。

Fig.1 Spatial-temporal expression patterns of NlGCHIa and NlGCHIb in Nilaparvata lugens (A) Relative expression levels of NlGCHIa and NlGCHIb during different developmental stages. N4-4h represents 4 h-old 4th-instar nymphs; FA-4h represents 4 h-old female adults; The other samples have the same naming method. (B) Relative expression levels of NlGCHIa and NlGCHIb in different tissues. MG, Midgut; FB, Fat body; CT, Cuticle; OV, Ovary; TE, Testis. The relative expression level was expressed as mean ±standard error (n=4).

图2 注射dsNlGCHI对褐飞虱NlGCHI mRNA表达水平和存活率的影响 (A)注射dsNlGCHI后NlGCHI基因的相对表达水平;(B)3龄若虫注射dsNlGCHI后的存活率曲线。*表示dsNlGCHI处理试虫的存活率显著低于dsGFP处理试虫。

Fig.2 Effects of dsNlGCHI injection on NlGCHI mRNA expression levels and survival rate in N. lugens (A) Relative expression level of NlGCHI post dsNlGCHI injection; (B) Survival pattern after injection of dsNlGCHI into 3rd instar nymphs. *, The survival rate of BPHs injected with dsNlGCHI is significantly lower than that injected with dsGFP.

图3 抑制NlGCHI mRNA表达水平对褐飞虱眼色、体色和死亡表型的影响 (A)注射dsNlGCHI后复眼颜色的表型;(B)注射dsNlGCHI后若虫不同时间段的体色变化;0 h、4 h和24 h分别表示初蜕皮、4 h和24 h日龄的5龄若虫;(C)注射dsNlGCHI后试虫死亡表型。

Fig.3 Effects of NlGCHI mRNA knockdown on eye coloration, body coloration and dead phenotype in N. lugens (A) The phenotype of compound eyes after dsNlGCHI injection; (B) The phenotype of body color after dsNlGCHI injection; 0 h, 4 h, and 24 h represent 0 h-, 4 h- and 24 h-old 5th-instar nymphs, respectively; (C) The dead phenotype after dsNlGCHI injection.

图4 抑制剂DAHP对褐飞虱生长发育的影响 (A)褐飞虱的存活率曲线;*表示抑制剂DAHP处理试虫的存活率显著低于对照组;(B)取食抑制剂DAHP后褐飞虱的死亡表型。

Fig.4 Effects of inhibitor DAHP on the growth and development of N. lugens (A) The survival rate curve of N. lugens; *, The survival rate of BPHs after feeding inhibitor DAHP is significantly lower than that in the control group; (B) The dead phenotype of N. lugens after feeding the inhibitor DAHP.

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GTP环化水解酶——一种防治褐飞虱(Nilaparvata lugens)的RNAi靶标基因

GTP cyclohydrolase: a RNAi target gene against the brown planthopper (Nilaparvata lugens)

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