Article(id=1266470561459880041, tenantId=1146029695717560320, journalId=1266358857061122103, issueId=1266470523241382909, articleNumber=null, orderNo=null, doi=10.13802/j.cnki.zwbhxb.2026.2025097, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1752595200000, receivedDateStr=2025-07-16, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1779879711737, onlineDateStr=2026-05-27, pubDate=1777478400000, pubDateStr=2026-04-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1779879711737, onlineIssueDateStr=2026-05-27, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1779879711737, creator=13701087609, updateTime=1779879711737, updator=13701087609, issue=Issue{id=1266470523241382909, tenantId=1146029695717560320, journalId=1266358857061122103, year='2026', volume='53', issue='2', pageStart='301', pageEnd='586', issueExtLink='null', onlineDate='null', pubDate='1777478400000', pubDateStr='2026-04-30', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1779879702622, creator='13701087609', updateTime=1782266106964, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276479828593349443, tenantId=1146029695717560320, journalId=1266358857061122103, issueId=1266470523241382909, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276479828593349444, tenantId=1146029695717560320, journalId=1266358857061122103, issueId=1266470523241382909, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=498, endPage=506, ext={EN=ArticleExt(id=1266470561740898412, articleId=1266470561459880041, tenantId=1146029695717560320, journalId=1266358857061122103, language=EN, title=Evaluation of the control efficacy of RNA interference targeting the FPPS gene against pest mites, columnId=1266470561661206635, journalTitle=Journal of Plant Protection, columnName=Research reports, runingTitle=null, highlight=null, articleAbstract=

To assess the feasibility of farnesyl pyrophosphate synthase (FPPS), a key enzyme in juvenile hormone biosynthesis, as a target for RNA interference (RNAi)-based control of pest mites, double-stranded RNA targeting FPPS (dsFPPS) was designed using the dsRNAEngineer online platform. The bioactivity of dsFPPS against two agriculturally important pest mites, Tetranychus urticae and Tetranychus evansi, was evaluated with microinjection. The expression levels of FPPS in mites after dsFPPS injection were determined by quantitative real-time PCR, and the safety to non-target organisms, Neoseiulus californicus and Harmonia axyridis, was assessed by feeding and microinjection assays. The results showed that after dsFPPS injection, FPPS transcript levels in T. urticae and T. evansi were significantly reduced by 91.80% and 83.09%, respectively. Deutonymphs of both mite species failed to molt normally and died, with mortality rates of 76.97% and 84.32%, respectively. After feeding on or microinjection dsFPPS, N. californicus and H. axyridis developed normally, indicating no significant effects on these natural enemies. These findings demonstrate that dsFPPS has high lethality against pest mites while being safe for non-target organisms. The FPPS gene can therefore serve as an ideal target for RNAi-based control of pest mites and has potential for development as a novel environmentally friendly acaricide.

, authors=null, authorsList=Gang Li, Qingyan Li, Zhuo Li, Zhangguang Cao, Hongwen Yu, Zhongyi Li, Xinyao Gu, authorCompany=null, correspAuthors=null, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1266470563036938359, articleId=1266470561459880041, tenantId=1146029695717560320, journalId=1266358857061122103, language=CN, title=靶向 FPPS 基因的RNA干扰技术对农业害螨的防控效果评估, columnId=1266470561837367405, journalTitle=植物保护学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

为明确保幼激素合成关键酶——法尼基焦磷酸合成酶(farnesyl pyrophosphate synthase,FPPS)作为害螨RNA干扰防控靶标的可行性,通过dsRNAEngineer在线平台设计FPPS的dsRNA(dsFPPS),采用注射法测定其对农业重要害螨二斑叶螨Tetranychus urticae和伊氏叶螨Tetranychus evansi的生物活性,采用实时荧光定量PCR技术检测注射dsFPPS 后害螨体内FPPS的表达水平,并通过饲喂法和注射法分别评价其对非靶标生物加州新小绥螨Neoseiulus californicus和异色瓢虫Harmonia axyridis的安全性。结果显示:注射dsFPPS后,二斑叶螨和伊氏叶螨体内FPPS的表达量分别显著降低了91.80%和83.09%;两种叶螨后若螨均无法正常蜕皮而死亡,死亡率分别为76.97%和84.32%。饲喂或注射dsFPPS后加州新小绥螨和异色瓢虫均发育正常,证明其对天敌生物无显著影响。表明dsFPPS对害螨具有高效致死性且对非靶标生物安全,FPPS基因可作为叶螨RNA干扰防控的理想靶标,具有开发为新型绿色杀螨剂的潜力。

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Pest Management Science, 74(6): 1239-1250, articleTitle=RNA interference technology in crop protection against arthropod pests, pathogens and nematodes, refAbstract=null)], funds=[Fund(id=1266746577566716012, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, awardId=GASTYESS202411, language=CN, fundingSource=贵州省科学技术协会青年科技人才托举工程项目(GASTYESS202411), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1266746565252239402, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, xref=1., ext=[AuthorCompanyExt(id=1266746565260628011, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, companyId=1266746565252239402, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.Institute of Entomology, Guizhou University, Guiyang 550025, Guizhou Province, China), AuthorCompanyExt(id=1266746565269016620, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, companyId=1266746565252239402, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.贵州大学昆虫研究所,贵阳 550025)]), AuthorCompany(id=1266746565344514093, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, xref=2., ext=[AuthorCompanyExt(id=1266746565352902702, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, companyId=1266746565344514093, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.College of Animal Science, Guizhou University, Guiyang 550025, Guizhou Province, China), AuthorCompanyExt(id=1266746565361291311, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, companyId=1266746565344514093, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.贵州大学动物科学学院,贵阳 550025)])], figs=[ArticleFig(id=1266746576165818466, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=EN, label=Fig. 1, caption=Distribution of on-target and off-target sites in the FPPS sequence, figureFileSmall=pWg9NZxy3dIJRH00FsbCiA==, figureFileBig=Ojqnws6JOKGNGHnSo9NOnQ==, tableContent=null), ArticleFig(id=1266746576505557091, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=CN, label=图1, caption=FPPS 基因序列在靶/脱靶位点分布图, figureFileSmall=pWg9NZxy3dIJRH00FsbCiA==, figureFileBig=Ojqnws6JOKGNGHnSo9NOnQ==, tableContent=null), ArticleFig(id=1266746576589443172, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=EN, label=Fig. 2, caption=Effect of dsFPPS injection on FPPS expression levels in Tetranychus urticae and Tetranychus evansi, figureFileSmall=P26MHjpKjMicEJH5pdo90g==, figureFileBig=zm8RMgoT7rDqaeYYDvMivA==, tableContent=null), ArticleFig(id=1266746576648163429, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=CN, label=图2, caption=注射dsFPPS 对二斑叶螨和伊氏叶螨体内 FPPS 基因表达水平的影响

图中数据为平均数±标准误。***表示对照与处理间经独立样本t检验法检验差异显著(P<0.001)。Data are mean±SE. *** indicates significant difference between the control and treatment groups based on an independent samples t test (P<0.001).

, figureFileSmall=P26MHjpKjMicEJH5pdo90g==, figureFileBig=zm8RMgoT7rDqaeYYDvMivA==, tableContent=null), ArticleFig(id=1266746576912404582, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=EN, label=Fig. 3, caption=Effect of dsFPPS injection on the development of deutonymphs of Tetranychus urticae and Tetranychus evansi, figureFileSmall=RM/4zzGr7bs3ILr9iL1qOg==, figureFileBig=hGVR84WAD0RuJ/8jzkzKQA==, tableContent=null), ArticleFig(id=1266746576996290663, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=CN, label=图3, caption=注射dsFPPS 对二斑叶螨和伊氏叶螨后若螨生长发育的影响

A:注射dsFPPS或dsGFP后两种叶螨的不同表型;B:注射dsFPPS或dsGFP后两种叶螨后若螨的发育情况。A: Phenotypic differences of mites after injection of dsFPPS or dsGFP; B: development of deutonymphs after injection of dsFPPS or dsGFP.

, figureFileSmall=RM/4zzGr7bs3ILr9iL1qOg==, figureFileBig=hGVR84WAD0RuJ/8jzkzKQA==, tableContent=null), ArticleFig(id=1266746577071788136, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=EN, label=Fig. 4, caption=Effect of dsFPPS on molting in Neoseiulus californicus and emergence in Harmonia axyridis, figureFileSmall=mwF+GcXcq4DVl1jUvxBhTg==, figureFileBig=Sg752jmb3wljeW0AvcIn8A==, tableContent=null), ArticleFig(id=1266746577323446377, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=CN, label=图4, caption=dsFPPS 对加州新小绥螨蜕皮和异色瓢虫羽化的影响

A~B:饲喂/注射dsFPPS后加州新小绥螨和异色瓢虫的表型;C~D:饲喂/注射dsFPPS或dsGFP后加州新小绥螨的蜕皮率和异色瓢虫的羽化率。A-B: Phenotypes of N. californicus and H. axyridis after feeding or injection with dsFPPS; C-D: molting rate of N. californicus and emergence rate of H. axyridis after feeding or injection with dsFPPS or dsGFP.

图中数据为平均数±标准误。***表示对照与处理间经独立样本t检验法检验差异显著(P<0.001)。Data are mean±SE. *** indicates a significant difference between the control and treatment groups based on an independent samples t test (P<0.001).

, figureFileSmall=mwF+GcXcq4DVl1jUvxBhTg==, figureFileBig=Sg752jmb3wljeW0AvcIn8A==, tableContent=null), ArticleFig(id=1266746577398943850, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=EN, label=Table 1, caption=

Primer information used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=

名称

Name

引物序列(5′-3′)

Primer sequence (5′-3′)

用途

Purpose

dsFPPSF: taatacgactcactatagggATGAACCAAAAATAAATATTGG

克隆dsRNA

Cloning of dsRNA

R: taatacgactcactatagggTTATTTACTTCGGCGGTAGA
dsGFPF: taatacgactcactatagggTGGGCACAAATTTTCTGTC
R: taatacgactcactatagggAAGGGTATCACCTTCAAAC
TuFPPSF: CTGCCCATTCGATTGGCTTT

实时荧光定量PCR

Real-time quantitative PCR

R: TCTCGGCCAATCTTTCCGAT
TuATPF: CCCGAAGAGATGATCCAAACTG
R: CGGTAAACCTGATGCTGAGAAA
TeFPPSF: TCTCTGACTTTGGCGACACT
R: GGACCCTGCTGTAATCGGAA
TeATPF: GTTTTCGCTGCAATGGGTGT
R: GTGTCGACCACATTGGTAAGC
), ArticleFig(id=1266746577453469803, tenantId=1146029695717560320, journalId=1266358857061122103, articleId=1266470561459880041, language=CN, label=表1, caption=

本研究所用引物信息

, figureFileSmall=null, figureFileBig=null, tableContent=

名称

Name

引物序列(5′-3′)

Primer sequence (5′-3′)

用途

Purpose

dsFPPSF: taatacgactcactatagggATGAACCAAAAATAAATATTGG

克隆dsRNA

Cloning of dsRNA

R: taatacgactcactatagggTTATTTACTTCGGCGGTAGA
dsGFPF: taatacgactcactatagggTGGGCACAAATTTTCTGTC
R: taatacgactcactatagggAAGGGTATCACCTTCAAAC
TuFPPSF: CTGCCCATTCGATTGGCTTT

实时荧光定量PCR

Real-time quantitative PCR

R: TCTCGGCCAATCTTTCCGAT
TuATPF: CCCGAAGAGATGATCCAAACTG
R: CGGTAAACCTGATGCTGAGAAA
TeFPPSF: TCTCTGACTTTGGCGACACT
R: GGACCCTGCTGTAATCGGAA
TeATPF: GTTTTCGCTGCAATGGGTGT
R: GTGTCGACCACATTGGTAAGC
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靶向 FPPS 基因的RNA干扰技术对农业害螨的防控效果评估
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李刚 1 , 李庆艳 1 , 李卓 1 , 曹璋光 1 , 于洪文 1 , 李忠意 2 , 古欣瑶 2
植物保护学报 | 研究论文 2026,53(2): 498-506
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植物保护学报 |研究论文 2026 , 53 (2) : 498 -506
靶向 FPPS 基因的RNA干扰技术对农业害螨的防控效果评估
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李刚1, 李庆艳1, 李卓1, 曹璋光1, 于洪文1, 李忠意2, 古欣瑶2
作者信息
  • 1.贵州大学昆虫研究所,贵阳 550025
  • 2.贵州大学动物科学学院,贵阳 550025
通讯作者:
Evaluation of the control efficacy of RNA interference targeting the FPPS gene against pest mites
Gang Li1, Qingyan Li1, Zhuo Li1, Zhangguang Cao1, Hongwen Yu1, Zhongyi Li2, Xinyao Gu2
Affiliations
  • 1.Institute of Entomology, Guizhou University, Guiyang 550025, Guizhou Province, China
  • 2.College of Animal Science, Guizhou University, Guiyang 550025, Guizhou Province, China
出版时间: 2026-04-30 doi: 10.13802/j.cnki.zwbhxb.2026.2025097
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为明确保幼激素合成关键酶——法尼基焦磷酸合成酶(farnesyl pyrophosphate synthase,FPPS)作为害螨RNA干扰防控靶标的可行性,通过dsRNAEngineer在线平台设计FPPS的dsRNA(dsFPPS),采用注射法测定其对农业重要害螨二斑叶螨Tetranychus urticae和伊氏叶螨Tetranychus evansi的生物活性,采用实时荧光定量PCR技术检测注射dsFPPS 后害螨体内FPPS的表达水平,并通过饲喂法和注射法分别评价其对非靶标生物加州新小绥螨Neoseiulus californicus和异色瓢虫Harmonia axyridis的安全性。结果显示:注射dsFPPS后,二斑叶螨和伊氏叶螨体内FPPS的表达量分别显著降低了91.80%和83.09%;两种叶螨后若螨均无法正常蜕皮而死亡,死亡率分别为76.97%和84.32%。饲喂或注射dsFPPS后加州新小绥螨和异色瓢虫均发育正常,证明其对天敌生物无显著影响。表明dsFPPS对害螨具有高效致死性且对非靶标生物安全,FPPS基因可作为叶螨RNA干扰防控的理想靶标,具有开发为新型绿色杀螨剂的潜力。

叶螨  /  RNA干扰  /  法尼基焦磷酸合成酶  /  特异性dsRNA  /  生物安全  /  防控靶标

To assess the feasibility of farnesyl pyrophosphate synthase (FPPS), a key enzyme in juvenile hormone biosynthesis, as a target for RNA interference (RNAi)-based control of pest mites, double-stranded RNA targeting FPPS (dsFPPS) was designed using the dsRNAEngineer online platform. The bioactivity of dsFPPS against two agriculturally important pest mites, Tetranychus urticae and Tetranychus evansi, was evaluated with microinjection. The expression levels of FPPS in mites after dsFPPS injection were determined by quantitative real-time PCR, and the safety to non-target organisms, Neoseiulus californicus and Harmonia axyridis, was assessed by feeding and microinjection assays. The results showed that after dsFPPS injection, FPPS transcript levels in T. urticae and T. evansi were significantly reduced by 91.80% and 83.09%, respectively. Deutonymphs of both mite species failed to molt normally and died, with mortality rates of 76.97% and 84.32%, respectively. After feeding on or microinjection dsFPPS, N. californicus and H. axyridis developed normally, indicating no significant effects on these natural enemies. These findings demonstrate that dsFPPS has high lethality against pest mites while being safe for non-target organisms. The FPPS gene can therefore serve as an ideal target for RNAi-based control of pest mites and has potential for development as a novel environmentally friendly acaricide.

spider mites  /  RNA interference  /  farnesyl pyrophosphate synthetase  /  specific dsRNA  /  biosafety  /  control target
李刚, 李庆艳, 李卓, 曹璋光, 于洪文, 李忠意, 古欣瑶. 靶向 FPPS 基因的RNA干扰技术对农业害螨的防控效果评估. 植物保护学报, 2026 , 53 (2) : 498 -506 . DOI: 10.13802/j.cnki.zwbhxb.2026.2025097
Gang Li, Qingyan Li, Zhuo Li, Zhangguang Cao, Hongwen Yu, Zhongyi Li, Xinyao Gu. Evaluation of the control efficacy of RNA interference targeting the FPPS gene against pest mites[J]. Journal of Plant Protection, 2026 , 53 (2) : 498 -506 . DOI: 10.13802/j.cnki.zwbhxb.2026.2025097
叶螨是重要的世界性经济害螨,寄主范围广,可取食为害蔬菜、果树和花卉等3 800多种寄主植物,在全球范围内造成了巨大的经济损失(Migeon et al.,2010;常芸等,2020)。化学防治是叶螨田间防控的主要手段,而频繁使用化学药剂导致叶螨抗药性问题十分突出(徐丹丹等,2018王帅宇等,2023董瑞等,2024)。农业重要害螨二斑叶螨Tetranychus urticae的田间抗药性监测结果显示,其对杀螨剂甲氰菊酯、溴虫腈以及阿维菌素的抗性倍数分别高达280.51倍、952.00倍和1 000.00倍以上(沈慧敏和杨宝生 2001徐丹丹,2019田甜等,2023);据节肢动物抗药性数据库统计,二斑叶螨已经成为世界上抗药性最严重的节肢动物之一。因此,发掘叶螨防控新靶标,开发特异性绿色防控技术体系,对叶螨田间抗药性治理有着重要的科学意义。RNA干扰(RNA interference,RNAi)技术通过引入外源dsRNA对靶标基因mRNA实现转录后沉默,从而出现表型缺陷或功能丧失。目前,利用RNAi技术抑制害虫生长发育和侵染为害的关键基因表达,可以阻碍其正常的生长、发育和繁殖,甚至导致死亡,具有低剂量、高防效和可持续的特点(Zhu & Palli,2020Silver et al.,2021Wang et al.,2024),已广泛用于马铃薯甲虫Leptinotarsa decemlineata、玉米根甲虫Diabrotica virgifera和黄曲条跳甲Phyllotreta striolata等农业害虫的防控(Zotti et al.,2018Yan et al.,2021)。因此,利用RNAi技术抑制叶螨生长发育过程关键基因表达,阻断其生长发育过程,可作为叶螨绿色防控技术开发新方向。
昆虫生长发育过程受到多种激素的相互协调作用,保幼激素(juvenile hormone,JH)作为昆虫生长发育过程的重要调控激素,决定了昆虫生长发育过程的形态和生理特征。JH的合成受到一系列合成酶的催化,法尼基焦磷酸合成酶(farnesyl pyrophosphate synthetase,FPPS)作为JH合成通路的关键酶之一,属于异戊二烯转移酶E家族中的碳链延伸酶,能够催化甲羟戊酸途径的最终反应,生成JH的前体法尼基焦磷酸(Dhar et al.,2013)。在昆虫体内,FPPS参与调控生长发育和蜕皮过程。例如在棉蚜Aphis gossypii中,利用RNAi技术抑制其3龄幼虫FPPS的表达后,蜕皮后的雌蚜体型明显变小,繁殖力显著下降(Sun & Li,2018);在家蚕Bombyx mori中,敲低BmFPPS会使JH滴度降低,导致幼虫出现性早熟现象(Fang et al.,2024);在棉铃虫Helicoverpa armigera中,敲低HaFpps4导致JH滴度降低,不仅对JH通路中其他基因的转录水平产生负面影响,还会使幼虫蜕皮受阻,最终死亡(Zhang et al.,2017)。在二斑叶螨中,利用RNAi技术抑制TuFPPS表达后,若螨出现发育延迟和蜕皮异常致死的现象(Li et al.,2025),表明FPPS在二斑叶螨变态发育过程发挥着重要作用。然而,FPPS是否可作为叶螨RNAi防控靶标尚不明晰。
本研究利用dsRNAEngineer在线平台(https://dsrna-engineer.cn)(Chen et al.,2025),根据二斑叶螨FPPS基因mRNA序列设计dsRNA(dsFPPS)片段,选用二斑叶螨和伊氏叶螨Tetranychus evansi进行RNAi试验,以评估dsFPPS对两种叶螨的防控效果;同时,对加州新小绥螨Neoseiulus californicus和异色瓢虫Harmonia axyridis进行RNAi试验,以评估dsFPPS对非靶标生物的安全性,探究FPPS作为叶螨RNAi防控靶标的潜力,以期为叶螨的新型绿色防控策略提供理论依据。
供试螨类和昆虫:二斑叶螨和伊氏叶螨均为贵州大学昆虫研究所保存品系,在室内分别以菜豆Phaseolus vulgaris(品种为紫花芸豆)和番茄Solanum lycopersicum(品种为中蔬4号)叶片饲养(Wang et al.,2023),取后若螨供试。加州新小绥螨购买于福州冠农生物科技有限公司,用二斑叶螨、伊氏叶螨和加州新小绥螨于温度(27±1) ℃、光照周期14 L∶10 D、相对湿度(65±5)%条件下饲养,取后若螨供试。异色瓢虫购买于河南省济源白云实业有限公司,用人工饲料(Sun et al.,2018)饲养,饲养温度和相对湿度同上,光照周期为10 L∶14 D,取4龄幼虫供试。
供试植物:紫花芸豆和中蔬4号番茄种子分别购自可米庄园商贸和金种子农资公司,在温度(27±1) ℃、光照周期14 L∶10 D、相对湿度(65±5)%条件下培养至幼苗期后,用于饲喂二斑叶螨和伊氏叶螨。
试剂:总RNA提取试剂(TRIzol),生工生物工程(上海)股份有限公司;胶回收试剂盒、dsRNA合成试剂盒,美国赛默飞世尔科技;cDNA反转录试剂盒、2×Taq PCR StarMix with Loading Dye、RealStar Green Fast Mixture试剂盒,北京康润诚业生物科技有限公司;其余试剂均为国产分析纯。
仪器:CFX96TM荧光定量PCR仪、C1000TM Thermal Cycler PCR仪、PowerPac Basic 电泳仪,伯乐生命医学产品(上海)有限公司;Nanodrop 2000超微量分光光度计,美国赛默飞世尔科技公司;SYS-PV830气动皮升操作泵、Micro-2T微量注射泵,世界精密仪器商贸(上海)有限公司;VHX-6000超景深三维显微镜,基恩士(中国)有限公司;RXZ-260B型智能型人工气候箱,宁波东南仪器有限公司。
在dsRNAEngineer在线平台输入二斑叶螨FPPS基因(命名为TuFPPS)的mRNA序列,选择二斑叶螨、伊氏叶螨、朱砂叶螨Tetranychus cinnabarinus、柑橘全爪螨Panonychus citri、截形叶螨Tetranychus truncatus和苹果全爪螨Panonychus ulmi为靶标物种,选择西方静走螨Galendromus occidentalis、巴氏新小绥螨Neoseiulus barkeri、加州新小绥螨和异色瓢虫为脱靶物种,完成叶螨在靶位点和非靶标生物脱靶位点分析后,将筛选出来的脱靶序列(以捕食螨的脱靶序列为主)用SnapGene软件剪切掉,将剩下的序列拼接起来,将以上步骤重复3次,把捕食螨所有的脱靶序列剪切后得到dsRNA片段并命名为dsFPPS。将该dsRNA片段的5′端添加T7启动子序列(taatacgactcactataggg)后,送至生工生物工程(上海)股份有限公司合成dsFPPS cDNA序列。
根据dsFPPS核酸序列,利用NCBI在线网站(https://www.ncbi.nlm.nih.gov/tools/primer-blast)设计并克隆该片段的引物(表1),本研究所有引物均由生工生物工程(上海)股份有限公司合成。以合成的dsFPPS cDNA序列为模板,通过特异性PCR扩增获得带有T7启动子序列的DNA片段,25 μL反应体系为:2×Taq PCR StarMix with Loading Dye 12.5 μL、上下游引物各1.0 μL、cDNA模板2.0 μL、DEPC-H2O 8.5 μL。然后以合成的带有T7启动子序列的DNA片段为模板,利用dsRNA合成试剂盒按照说明书合成dsRNA并纯化。
采用注射法评估dsFPPS对二斑叶螨和伊氏叶螨的防控效果。将1.2.1纯化后的dsFPPS浓度调整至10 000 ng/μL,使用气动皮升操作泵将dsRNA注射入刚蜕皮8 h以内的二斑叶螨和伊氏叶螨后若螨体内,每头螨大约注射1~2 nL的dsFPPS,以注射等量绿色荧光蛋白(green fluorescent protein,GFP)基因的dsRNA(dsGFP)为对照,制备方法同1.2.1。每个处理4个重复,每个重复至少25头螨(Wei et al.,2021Li et al.,2024)。将注射后的二斑叶螨和伊氏叶螨分别挑回菜豆叶和番茄叶上继续饲养,饲养条件同1.1。每12 h观察并记录螨虫生长发育情况以及蜕皮和死亡的数量,计算蜕皮率和死亡率,并用超景深三维显微镜成像系统进行拍照,直至所有螨虫完成蜕皮或者死亡。统计后若螨在不同时间点的发育情况(后若螨活动期、静止期和成螨)和死亡数量。
选取二斑叶螨和伊氏叶螨刚蜕皮8 h的后若螨,分别注射10 000 ng/μL的dsFPPS和dsGFP后24 h收集样品,每头螨大约注射1~2 nL,每个处理4个生物学重复,每个重复40头后若螨。利用总RNA提取试剂(TRIzol)提取样品的总RNA,用超微量分光光度计测定RNA的浓度和质量,并用1%琼脂糖凝胶检测RNA的完整性。取检测合格的总RNA,参照cDNA反转录试剂盒说明书合成cDNA模板,于-20 ℃保存备用。根据二斑叶螨和伊氏叶螨FPPS基因(TuFPPSTeFPPS)和内参基因ATPTuATPTeATP)的开放阅读框序列,利用NCBI网站设计实时荧光定量PCR引物(表1)。以分别注射dsFPPS和dsGFP后的二斑叶螨和伊氏叶螨的cDNA为模板,利用上述引物和RealStar Green Fast Mixture试剂盒进行目的基因的检测,分析二斑叶螨和伊氏叶螨体内FPPS的沉默效率。10 μL实时荧光定量PCR反应体系:2×RealStar Green Fast Mixture 5.0 μL、cDNA模板4.0 μL、上下游引物各0.5 μL。每个样品设置2个技术重复。采用2-ΔΔCt 法计算FPPS在二斑叶螨和伊氏叶螨中的相对表达量,计算FPPS的沉默效率。沉默效率=(对照相对表达量-处理相对表达量)/对照相对表达量×100%。
采用液滴饲喂法评估dsFPPS对加州新小绥螨的安全性。试验设dsFPPS处理组和dsGFP对照组,将dsFPPS和dsGFP的浓度均调整至10 000 ng/μL,分别取二者的dsRNA 7.4 μL、饱和蔗糖溶液2 μL和蓝色可食用色素染料0.06 μL制备成10 μL人工液体饲料(Zhu et al.,2024)。将刚蜕皮12 h内的加州新小绥螨后若螨挑至2 cm×2 cm的菜豆叶碟上饥饿24 h,再将存活加州新小绥螨挑至新的叶碟上;每个叶碟分别滴加10 μL上述制备好的含有dsFPPS或dsGFP的人工液体饲料。每个处理4个生物学重复,每个重复至少20头。然后将处理后的加州新小绥螨置于智能型人工气候箱中饲养12 h,饲养条件同1.1。取体色变蓝的加州新小绥螨用于后续试验,饲喂dsRNA 12 h后用二斑叶螨继续喂养,每隔12 h观察并记录一次试螨的生长发育情况以及蜕皮和死亡的数量,计算蜕皮率和死亡率,并用超景深三维显微镜成像系统进行拍照,直至所有螨虫完成蜕皮或死亡。
采用注射法评估dsFPPS对异色瓢虫的安全性。将dsFPPS和dsGFP的浓度均调整至10 000 ng/μL,用Micro-2T微量注射泵从刚蜕皮1 d内的异色瓢虫4龄幼虫腹部节间膜向其体内注射250 nL的dsFPPS或dsGFP,每个处理4个生物学重复,每个重复5头幼虫(Zhang et al.,2021)。注射后的异色瓢虫幼虫在直径为35 mm的培养皿中单头饲养,并提供1个浸透水的脱脂棉球补充水分,将培养皿放在智能型人工气候箱中用人工饲料继续饲养,条件同1.1。每天换一次饲料和供水棉球,观察幼虫的存活情况,记录存活数、化蛹数和羽化数,并用超景深三维显微镜成像系统进行拍照,直至所有虫体死亡或羽化为成虫为止。统计异色瓢虫4龄幼虫在不同发育时间点的蜕皮数量、死亡数量和羽化数量,计算蜕皮率、死亡率和羽化率。
采用Excel 2021对原始数据进行统计,用SPSS 22.0软件应用独立样本t检验法对RNAi处理后对照组和处理组蜕皮率或羽化率进行差异显著性分析,用GraphPad Prism 8.0.1软件绘图。
通过dsRNAEngineer在线平台对二斑叶螨TuFPPS基因mRNA序列进行评估,获得长度为590 bp的dsFPPS。dsFPPS共有1 552个害螨在靶位点,97个非靶标生物脱靶位点(图1)。在害螨中,二斑叶螨有680个在靶位点,伊氏叶螨有147个在靶位点,朱砂叶螨有374个在靶位点,柑橘全爪螨有95个在靶位点,截形叶螨有187个在靶位点,苹果全爪螨有73个在靶位点。在非靶标生物中,捕食螨中的西方静走螨、巴氏新小绥螨和加州新小绥螨均未发现脱靶位点,异色瓢虫有97个脱靶位点。
注射dsFPPS后,二斑叶螨和伊氏叶螨体内FPPS的表达量分别显著降低了91.80%和83.09%(图2)。两种叶螨注射dsFPPS后均出现了后若螨因无法正常完成蜕皮而被困在旧表皮中死亡的致死表型,注射dsFPPS后108 h二斑叶螨和伊氏叶螨的死亡率分别为76.97%和84.32%;而注射dsGFP对照的二斑叶螨和伊氏叶螨的死亡率分别为6.00%和5.76%(图3-A)。
观察注射dsFPPS的二斑叶螨和伊氏叶螨后若螨的生长发育情况,注射后24 h时二斑叶螨和伊氏叶螨后若螨进入静息状态的占比分别有19.74%和16.67%,48 h时二斑叶螨和伊氏叶螨的蜕皮率分别为1.32%和3.92%,二者最终的蜕皮率分别为23.03%和15.69%;而在dsGFP对照组中,24 h时二斑叶螨和伊氏叶螨后若螨进入静息状态的占比分别有54.00%和18.71%,48 h时二斑叶螨和伊氏叶螨的蜕皮率分别为58.67%和4.32%,二者最终的蜕皮率分别为94.00%和94.24%(图3-B)。表明注射 dsFPPS后,二斑叶螨和伊氏叶螨均出现不能正常蜕皮而死亡的现象。
饲喂dsFPPS后,加州新小绥螨后若螨能正常蜕皮发育为成螨(图4-A);注射dsFPPS后,异色瓢虫4龄幼虫可以正常化蛹并羽化为成虫(图4-B)。对加州新小绥螨蜕皮情况和异色瓢虫羽化情况进行统计分析,饲喂dsFPPS后加州新小绥螨后若螨最终的蜕皮率为96.27%,饲喂dsGFP的对照组最终的蜕皮率为97.56%,二者之间无显著差异,但在饲喂后48~72 h期间,dsGFP对照组的加州新小绥螨后若螨蜕皮率显著高于dsFPPS处理组,其余时间点处理组与对照组的蜕皮率均无显著差异(图4-C)。注射dsFPPS后异色瓢虫最终的羽化率为90.00%,注射dsGFP后异色瓢虫最终的羽化率为95.00%,二者之间无显著差异(图4-D)。
RNAi技术凭借环境安全性高、兼容性强的优势,成为一种新颖的防控技术并融入有害生物综合治理策略,符合经济、社会和农业生产对于绿色农药的需求(Zhang et al.,2013Leggewie & Schnettler,2018Wang et al.,2021)。RNAi通过特异性靶向害虫物种中的关键基因,能够影响害虫的存活、发育和繁殖等生理过程(Guo et al.,2021Lu & Shen,2024)。基于RNAi技术的害虫防控策略在有害生物综合防控中显示出巨大的发展潜力,然而在害螨防控中则少有报道(Baum et al.,2007Zhang et al.,2013Leggewie & Schnettler,2018)。前期研究发现,利用RNAi技术抑制二斑叶螨JH合成通路基因TuFPPS的表达,能够阻断二斑叶螨的生长发育过程,说明JH合成基因TuFPPS可作为防控二斑叶螨的RNAi靶标(Li et al.,2025)。因此,本研究在此基础上,利用dsRNAEngineer在线平台设计dsRNA,评估了FPPS基因在叶螨中高度保守的在靶位点,但在非靶标生物中的同源性较低,以此设计了叶螨特异性dsRNA序列,从而降低了RNAi对非靶标天敌的脱靶风险。
筛选高效且安全的RNAi靶标是开发绿色防控技术的基础。本研究利用RNAi技术评价了FPPS基因的防控效果和生物安全性,结果表明dsFPPS能有效抑制二斑叶螨和伊氏叶螨的生长发育过程,造成叶螨蜕皮畸形进而大量死亡,且对非靶标生物加州新小绥螨和异色瓢虫均无显著影响。另外,对叶螨基因组的分析发现,叶螨中仅存在单个FPPS基因,在JH合成通路中无其他功能冗余或同源基因,这一特点使其在单基因靶向RNAi时FPPS基因不易被其他同功能基因或同源基因替代,能提高叶螨RNAi的稳定性。针对单基因靶向RNAi策略中普遍存在的潜在风险,如与非靶标基因部分同源导致非特异性沉默(Lundgren & Duan,2013)、长期使用可能诱导靶标基因突变(He et al.,2020),以及因基因家族功能冗余导致的RNAi效果减弱(Wu et al.,2022Zhang et al.,2024),本研究在dsRNA设计阶段进行了针对性优化。首先,通过序列比对,去除与非靶标生物加州新小绥螨和异色瓢虫基因组同源性较高的保守片段,保留了叶螨特异性序列,从而有效降低了非特异性沉默风险。其次,FPPS基因在叶螨基因组中为单拷贝,且JH合成通路中无其他功能冗余基因,这一特性使其天然规避了基因代偿问题,进一步保障了RNAi的稳定性。综上,经序列优化的FPPS基因dsRNA兼具高效性、特异性和安全性,可作为叶螨绿色防控RNA生物农药的候选靶标。
本研究采用显微注射法作为RNAi递送方式,具有高效率和强稳定性的特点,但受限于操作复杂性,难以适用于田间规模化防控。因此,开发适用于田间环境的RNAi递送技术是提升RNAi田间防控效率的关键。目前,dsRNA常用递送方式主要包括显微注射、饲喂、喷洒、浸泡和点滴等。前期研究通过系统比较叶螨中不同递送方式的RNAi效率,发现浸泡法和叶片饲喂法是最有效的两种方式,有望成为未来田间喷雾型dsRNA产品的核心递送方案(Suzuki et al.,2017)。此外,其他递送策略也取得显著进展,例如表达细胞核/细胞质靶向dsRNA的转基因植物对朱砂叶螨具有致死效应(Wu et al.,2023);而利用RNase缺陷型大肠杆菌Escherichia coli HT115菌株等微生物载体合成递送dsRNA,在试验中也表现出良好的RNAi效果(Cedeño et al.,2015)。这些递送策略也应给予更多关注。
  • 贵州省科学技术协会青年科技人才托举工程项目(GASTYESS202411)
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2026年第53卷第2期
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doi: 10.13802/j.cnki.zwbhxb.2026.2025097
  • 接收时间:2025-07-16
  • 首发时间:2026-05-27
  • 出版时间:2026-04-30
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  • 收稿日期:2025-07-16
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贵州省科学技术协会青年科技人才托举工程项目(GASTYESS202411)
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    1.贵州大学昆虫研究所,贵阳 550025
    2.贵州大学动物科学学院,贵阳 550025

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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