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Article(id=1226236831621103856, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226236828399878330, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240781, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1733241600000, receivedDateStr=2024-12-04, revisedDate=null, revisedDateStr=null, acceptedDate=1735574400000, acceptedDateStr=2024-12-31, onlineDate=1770287243037, onlineDateStr=2026-02-05, pubDate=1746288000000, pubDateStr=2025-05-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770287243037, onlineIssueDateStr=2026-02-05, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770287243037, creator=13701087609, updateTime=1770287243037, updator=13701087609, issue=Issue{id=1226236828399878330, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='5', pageStart='1831', pageEnd='2319', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770287242269, creator=13701087609, updateTime=1770344517883, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226477059812274835, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226236828399878330, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226477059816469140, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226236828399878330, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2240, endPage=2251, ext={EN=ArticleExt(id=1226236834288681273, articleId=1226236831621103856, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Recombinant expression of shRNA targeting gat in microalgae for controlling Aedes albopictus, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To explore the technical feasibility of using transgenic microalgae to control Aedes that transmit diseases such as dengue fever. [Methods] Taking the neurotransmitter transporter gamma-aminobutyric acid receptor gene (gat) of Aedes as a target, we constructed a shRNA-expressing vector and then transferred the vector into Chlamydomonas reinhardtiii CC124 and Chlorella vulgaris HOC5 through electroporation. The recombinant microalgae were used to feed the larvae and adults of Aedes albopictus. [Results] The transgenic algae delayed the larval development and had obvious lethal effects on the larvae and adults of A. albopictus, causing the mortality rates of 75.56% and 58.67%, respectively. The expression level of gat in the larvae was significantly down-regulated. [Conclusion] Deploying gat-shRNA transgenic microalgae in enclosed water environments to suppress mosquito populations in the vicinity is technically feasible. This strategy provides a new perspective on using biological methods to control mosquitoes and block the transmission of severe infectious diseases such as dengue fever and Zika virus disease.

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E-mail: XUE Chunmei,
FEI Xiaowen,
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【目的】探讨利用转基因微藻防控传播登革热等疾病的伊蚊的技术可行性。【方法】以伊蚊神经递质转运体γ-氨基丁酸受体基因(gamma-aminobutyric acid receptor gene, gat)为靶点，构建shRNA微藻(Microalgae)表达载体，通过电击法将其转入莱茵衣藻(Chlamydomonas reinhardtii) CC124和小球藻(Chlorella vulgaris) HOC5中，获得重组微藻，并用其饲喂伊蚊幼虫和成蚊。【结果】gat-shRNA转基因藻株可显著延缓伊蚊幼虫的发育、对伊蚊幼虫和成蚊表现出明显的致死效果，致死率分别为75.56%和58.67%。伊蚊幼虫体内目标基因gat的表达量显著降低。【结论】在封闭水域中投放gat-shRNA转基因微藻以抑制水域附近的蚊子种群是可行的。该策略为使用生物方法控制伊蚊，以及阻断登革热、寨卡病毒病等严重传染病的传播提供了一种新的思路。

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作者贡献声明

唐欣欣：实验实施，结果分析，论文写作与修改；邓晓东：实验构思及设计，技术支持；黄小丹：实验实施；杨思琪：论文讨论及数据整理；薛春梅：论文讨论及修改；费小雯：实验构思及设计。

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International Journal of Environmental Research and Public Health, 2017, 14(4): 398., articleTitle=Attractive toxic sugar bait (ATSB) for control of mosquitoes and its impact on non-target organisms: a review, refAbstract=null)], funds=[Fund(id=1226592761101599278, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, awardId=ZDYF2022SHFZ314, language=EN, fundingSource=Key Research and Development Program of Hainan Province(ZDYF2022SHFZ314), fundOrder=null, country=null), Fund(id=1226592761227428406, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, awardId=ZDYF2022SHFZ314, language=CN, fundingSource=海南省重点研发计划(ZDYF2022SHFZ314), fundOrder=null, country=null), Fund(id=1226592761382617662, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, awardId=82260669, language=EN, fundingSource=National Natural Science Foundation of China(82260669), fundOrder=null, country=null), 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articleId=1226236831621103856, xref=4., ext=[AuthorCompanyExt(id=1226592752587161721, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, companyId=1226592752578773111, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4.Zhanjiang Experimental Station, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, Guangdong, China), AuthorCompanyExt(id=1226592752599744634, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, companyId=1226592752578773111, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4.中国热带农业科学院湛江实验站，广东 湛江)])], figs=[ArticleFig(id=1226592757779710395, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Figure 1, caption=Agarose gel electrophoresis. A: gat-shRNA secondary structure prediction diagram; B: Identification result of gat-p1302 (Lane M: DL5000 DNA marker; Lanes 1-2: gat-p1302; Lane 3: pCAMBIA1302 plasmid); C: Transgenic algae identification result diagram (Lane M: DL500 DNA marker; Lanes 1-5: Transformants 1-5 of gat-shRNA Chlorella; Lanes 7-12: Transformants 1-5 of gat-shRNA C. reinhardtii; Lanes 14-15: Transformants 1-2 of C. reinhardtii with the empty vector pCAMBIA1302; Lanes 16-18: Transformants 1-2 of Chlorella with the empty vector pCAMBIA1302; Lanes 6 and 13: p1302-gat plasmid; Lane 19: CC124 DNA; Lane 20: pCAMBIA1302 plasmid)., figureFileSmall=fnluzwmLpDZSJfv2kbALWA==, figureFileBig=VXsqO1eeIzKOHioDhnh2Cw==, tableContent=null), ArticleFig(id=1226592757888762304, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=图1, caption=琼脂糖凝胶电泳图。A：gat-shRNA二级结构预测图；B：gat-p1302鉴定结果图(Lane M：DL5000 DNA marker；Lanes 1-2：gat-p1302；Lane 3：pCAMBIA1302质粒)；C：转基因藻鉴定结果图(Lane M：DL500 DNA marker；Lanes 1-5：gat-shRNA小球藻转化子1-5；Lanes 7-12：gat-shRNA莱茵衣藻转化子1-5；Lanes 14-15：转空载体pCAMBIA1302的莱茵衣藻转化子1-2；Lanes 16-18：转空载体pCAMBIA1302的小球藻转化子1-2；Lanes 6、13：p1302-gat质粒；Lane 19：CC124 DNA；Lane 20：pCAMBIA1302质粒)。, figureFileSmall=fnluzwmLpDZSJfv2kbALWA==, figureFileBig=VXsqO1eeIzKOHioDhnh2Cw==, tableContent=null), ArticleFig(id=1226592758056534475, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Figure 2, caption=Mortality rate of Aedes aegypti larvae fed with recombinant microalgae. A: Mortality rate of larvae fed with gat-shRNA transformed C. reinhardtii strains 1-5 (CC124: Larvae fed with wild-type C. reinhardtii); B: Mortality rate of larvae fed with gat-shRNA transformed Chlorella strains 1-5 (HOC5: Larvae fed with wild-type Chlorella). Fodder: Larvae fed with feed; p1302: Larvae fed with Chlorella transformed with the empty vector pCAMBIA1302; gat-p1302-1-gat-p1302-5: Larvae fed with Chlorella transformed with gat-shRNA., figureFileSmall=sYLE6jmiRz56MDa7BTUWIw==, figureFileBig=CJSSgg0N0G9mU4Jkn4o/1Q==, tableContent=null), ArticleFig(id=1226592759419683278, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=图2, caption=饲喂重组微藻的伊蚊幼虫死亡率。A：饲喂转莱茵衣藻gat-shRNA转化子1-5的幼虫死亡率(CC124：野生莱茵衣藻饲喂幼虫)；B：饲喂转小球藻gat-shRNA转化子1-5的幼虫死亡率(HOC5：野生小球藻饲喂幼虫)。Fodder：用饲料饲喂幼虫；p1302：转空载体pCAMBIA1302的小球藻饲喂幼虫；gat-p1302-1-gat-p1302-5：转gat-shRNA小球藻饲喂幼虫。, figureFileSmall=sYLE6jmiRz56MDa7BTUWIw==, figureFileBig=CJSSgg0N0G9mU4Jkn4o/1Q==, tableContent=null), ArticleFig(id=1226592759595844052, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Figure 3, caption=Length of Aedes aegypti larvae under the microscope. A: Larvae fed with transgenic Chlorella (HOC5: Larvae fed with wild-type Chlorella); B: Larvae fed with transgenic C. reinhardtii (CC124: Larvae fed with wild-type C. reinhardtii). Water: Larvae fed with filtered water; Fodder: Larvae fed with feed; p1302: Larvae fed with C. reinhardtii transformed with the empty vector pCAMBIA1302; gat-p1302: Larvae fed with C. reinhardtii transformed with gat-shRNA., figureFileSmall=SC6pzgJH4Ylz/qrcCtDvUA==, figureFileBig=bqE8BUpYIlbkc/t+LRKOuQ==, tableContent=null), ArticleFig(id=1226592759725867487, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=图3, caption=显微镜下伊蚊幼虫的体长。A：转基因小球藻饲喂伊蚊幼虫(HOC5：野生小球藻饲喂幼虫)；B：转基因莱茵衣藻饲喂伊蚊幼虫(CC124：野生莱茵衣藻饲喂幼虫)。Water：用过滤水饲喂幼虫；Fodder：用饲料饲喂幼虫；p1302：转空载体pCAMBIA1302的莱茵衣藻饲喂幼虫；gat-p1302：转gat-shRNA莱茵衣藻饲喂幼虫。, figureFileSmall=SC6pzgJH4Ylz/qrcCtDvUA==, figureFileBig=bqE8BUpYIlbkc/t+LRKOuQ==, tableContent=null), ArticleFig(id=1226592759860085223, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Figure 4, caption=Larval body length of Aedes aegypti after being fed different foods. A: Body length of larvae fed with gat-shRNA transformed C. reinhardtii strains 1-5; B: Body length of larvae fed with gat-shRNA transformed Chlorella strains 1-5. Water: Larvae fed with filtered water; Fodder: Larvae fed with feed; HOC5: Larvae fed with wild-type Chlorella; p1302: Larvae fed with Chlorella transformed with the empty vector pCAMBIA1302; gat-p1302-1-gat-p1302-5: Larvae fed with Chlorella transformed with gat-shRNA. Different small letters indicate significant difference (P<0.05)., figureFileSmall=9eCHYUaeqR+fIQsTviR8xw==, figureFileBig=yWBcmqtuKh3Ca1JpP1zWPg==, tableContent=null), ArticleFig(id=1226592759981720047, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=图4, caption=饲喂不同食物后伊蚊幼虫的体长。A：转gat-shRNA莱茵衣藻转化子1-5饲喂幼虫体长；B：转gat-shRNA小球藻转化子1-5喂饲幼虫体长。Water：用过滤水饲喂幼虫；Fodder：用饲料饲喂幼虫；HOC5：野生小球藻饲喂幼虫；p1302组：转空载体pCAMBIA1302的小球藻饲喂幼虫；gat-p1302-1-gat-p1302-5：转gat-shRNA小球藻饲喂幼虫。不同小写字母表示显著差异(P<0.05)。, figureFileSmall=9eCHYUaeqR+fIQsTviR8xw==, figureFileBig=yWBcmqtuKh3Ca1JpP1zWPg==, tableContent=null), ArticleFig(id=1226592760111743479, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Figure 5, caption=Target gene expression levels in Aedes aegypti larvae after being fed shRNA recombinant microalgae. Fodder: Larvae fed with feed; CC124: Larvae fed with wild-type C. reinhardtii; HOC5: Larvae fed with wild-type Chlorella; gat-CC124: Larvae fed with gat-shRNA C. reinhardtii; gat-HOC5: Larvae fed with gat-shRNA Chlorella. Different small letters indicate significant difference (P<0.05)., figureFileSmall=Mt3I63Dfhs0ZWECbfWuGrw==, figureFileBig=+9umkYkj4f9y6MTIE35nQg==, tableContent=null), ArticleFig(id=1226592760224989693, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=图5, caption=饲喂shRNA重组微藻后伊蚊幼虫靶基因表达量。Fodder：用饲料饲喂幼虫；CC124：野生莱茵衣藻饲喂幼虫；HOC5：野生小球藻饲喂幼虫；gat-CC124：gat-shRNA莱茵衣藻饲喂幼虫；gat-HOC5：gat-shRNA小球藻饲喂幼虫。不同小写字母表示显著差异(P<0.05)。, figureFileSmall=Mt3I63Dfhs0ZWECbfWuGrw==, figureFileBig=+9umkYkj4f9y6MTIE35nQg==, tableContent=null), ArticleFig(id=1226592760342430211, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Figure 6, caption=Lethal rate of transgenic microalgae on adult mosquitoes. CC124+10% sucrose: A mixture of wild-type CC124 and 10% sucrose; Toxic+10% sucrose: Mixture of 0.1 mg/L deltamethrin and 10% sucrose; gat-p1302+10% sucrose: gat-shRNA mixture of C. reinhardtii and 10% sucrose., figureFileSmall=PsL6xNe9hKFILXLKiBY3zw==, figureFileBig=jda49CoAehvldcsEFODoZg==, tableContent=null), ArticleFig(id=1226592760518590989, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=图6, caption=转基因微藻对成蚊的致死率。CC124+10% sucrose：野生CC124藻株和10%蔗糖的混合液；Toxic+10% sucrose：0.1 mg/L溴氰菊酯和10%蔗糖的混合液；gat-p1302+10% sucrose：gat-shRNA莱茵衣藻和10%蔗糖的混合液。, figureFileSmall=PsL6xNe9hKFILXLKiBY3zw==, figureFileBig=jda49CoAehvldcsEFODoZg==, tableContent=null), ArticleFig(id=1226592760615059988, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=EN, label=Table 1, caption=

Design of shRNA sequences, target gene amplification and internal reference primers

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer sequences (5′→3′)Initial position
Gat target sequenceCGTGTGCTTCATCAGTTATTT325
Gat-shRNA-F

GATCCGTGTGCTTCATCAGTTATTTCTCGAGAAATAA

CTGATGAAGCACACGTTTTTG

Gat-shRNA-R

GTCACCAAAAACGTGTGCTTCATCAGTTATTTCTCG

AGAAATAACTGATGAAGCACACG

Gat transcripts

GATCCGTGTGCTTCATCAGTTATTTCTCGAGAAA

TAACTGATGAAGCACACG

pCAMBIA1302-FTTACCCAACTTAATCGCCTTGCAG
pCAMBIA1302-RTATCGCAATGATGGCATTTGTAGG
Gat-FTGCCTTGTTCCCGTACTTCC
Gat-RGCGTTACGGCATCAATCCAC
PRS17-FAAGAAGTGGCCATCATTCCA
PRS17-RGGTCTCCGGGTCGACTTC
), ArticleFig(id=1226592760807997984, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236831621103856, language=CN, label=表1, caption=

shRNA序列、扩增靶基因及内参引物的设计

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer sequences (5′→3′)Initial position
Gat target sequenceCGTGTGCTTCATCAGTTATTT325
Gat-shRNA-F

GATCCGTGTGCTTCATCAGTTATTTCTCGAGAAATAA

CTGATGAAGCACACGTTTTTG

Gat-shRNA-R

GTCACCAAAAACGTGTGCTTCATCAGTTATTTCTCG

AGAAATAACTGATGAAGCACACG

Gat transcripts

GATCCGTGTGCTTCATCAGTTATTTCTCGAGAAA

TAACTGATGAAGCACACG

pCAMBIA1302-FTTACCCAACTTAATCGCCTTGCAG
pCAMBIA1302-RTATCGCAATGATGGCATTTGTAGG
Gat-FTGCCTTGTTCCCGTACTTCC
Gat-RGCGTTACGGCATCAATCCAC
PRS17-FAAGAAGTGGCCATCATTCCA
PRS17-RGGTCTCCGGGTCGACTTC
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靶向 gat 基因的shRNA重组微藻防控白纹伊蚊
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唐欣欣 1 , 邓晓东 2, 4 , 黄小丹 3 , 杨思琪 1 , 薛春梅 1 , 费小雯 3
微生物学报 | 研究报告 2025,65(5): 2240-2251
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微生物学报 | 研究报告 2025, 65(5): 2240-2251
靶向 gat 基因的shRNA重组微藻防控白纹伊蚊
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唐欣欣1, 邓晓东2, 4, 黄小丹3, 杨思琪1, 薛春梅1 , 费小雯3
作者信息
  • 1.佳木斯大学 生物与农业学院，黑龙江 佳木斯
  • 2.中国热带农业科学院热带生物技术研究所，海南省海洋生物资源功能性成分研究与利用重点实验室，海南 海口
  • 3.海南医科大学 基础医学与生命科学学院，海南 海口
  • 4.中国热带农业科学院湛江实验站，广东 湛江
Recombinant expression of shRNA targeting gat in microalgae for controlling Aedes albopictus
Xinxin TANG1, Xiaodong DENG2, 4, Xiaodan HUANG3, Siqi YANG1, Chunmei XUE1 , Xiaowen FEI3
Affiliations
  • 1.College of Biology and Agriculture, Jiamusi University, Jiamusi, Heilongjiang, China
  • 2.Hainan Provincial Key Laboratory for Functional Components Research and Utilization of Marine Bioresources in Hainan Province, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China
  • 3.School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou, Hainan, China
  • 4.Zhanjiang Experimental Station, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, Guangdong, China
出版时间: 2025-05-04 doi: 10.13343/j.cnki.wsxb.20240781
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摘要
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【目的】探讨利用转基因微藻防控传播登革热等疾病的伊蚊的技术可行性。【方法】以伊蚊神经递质转运体γ-氨基丁酸受体基因(gamma-aminobutyric acid receptor gene, gat)为靶点，构建shRNA微藻(Microalgae)表达载体，通过电击法将其转入莱茵衣藻(Chlamydomonas reinhardtii) CC124和小球藻(Chlorella vulgaris) HOC5中，获得重组微藻，并用其饲喂伊蚊幼虫和成蚊。【结果】gat-shRNA转基因藻株可显著延缓伊蚊幼虫的发育、对伊蚊幼虫和成蚊表现出明显的致死效果，致死率分别为75.56%和58.67%。伊蚊幼虫体内目标基因gat的表达量显著降低。【结论】在封闭水域中投放gat-shRNA转基因微藻以抑制水域附近的蚊子种群是可行的。该策略为使用生物方法控制伊蚊，以及阻断登革热、寨卡病毒病等严重传染病的传播提供了一种新的思路。

莱茵衣藻  /  小球藻  /  登革热  /  伊蚊  /  gat基因

[Objective] To explore the technical feasibility of using transgenic microalgae to control Aedes that transmit diseases such as dengue fever. [Methods] Taking the neurotransmitter transporter gamma-aminobutyric acid receptor gene (gat) of Aedes as a target, we constructed a shRNA-expressing vector and then transferred the vector into Chlamydomonas reinhardtiii CC124 and Chlorella vulgaris HOC5 through electroporation. The recombinant microalgae were used to feed the larvae and adults of Aedes albopictus. [Results] The transgenic algae delayed the larval development and had obvious lethal effects on the larvae and adults of A. albopictus, causing the mortality rates of 75.56% and 58.67%, respectively. The expression level of gat in the larvae was significantly down-regulated. [Conclusion] Deploying gat-shRNA transgenic microalgae in enclosed water environments to suppress mosquito populations in the vicinity is technically feasible. This strategy provides a new perspective on using biological methods to control mosquitoes and block the transmission of severe infectious diseases such as dengue fever and Zika virus disease.

Chlamydomonas reinhardtiii  /  Chlorella vulgaris  /  dengue fever  /  Aedes  /  gat
唐欣欣, 邓晓东, 黄小丹, 杨思琪, 薛春梅, 费小雯. 靶向 gat 基因的shRNA重组微藻防控白纹伊蚊. 微生物学报, 2025 , 65 (5) : 2240 -2251 . DOI: 10.13343/j.cnki.wsxb.20240781
Xinxin TANG, Xiaodong DENG, Xiaodan HUANG, Siqi YANG, Chunmei XUE, Xiaowen FEI. Recombinant expression of shRNA targeting gat in microalgae for controlling Aedes albopictus[J]. Acta Microbiologica Sinica, 2025 , 65 (5) : 2240 -2251 . DOI: 10.13343/j.cnki.wsxb.20240781
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登革热(dengue fever, DF)是一种由蚊子传播的疾病，已经成为全球范围内一个非常紧急和重要的公共卫生问题。根据世界卫生组织(World Health Organization, WHO)的统计数据(https://www.who.int/zh/emergencies/disease-outbreak-news/item/2023-DON498)，从2000年到2019年，全球报告的登革热病例数量从50万增加到了520万，这些病例分布在129个国家。2024年全球登革热疫情高发，多国出现病例数上升。据WHO通报(https://new.qq.com/rain/a/20240520A08ITS00)，美洲地区2024年1-3月报告的登革热病例数超过300万，是2023年的3倍；巴西发现的登革热疑似及确诊病例累计已超过427万例，死亡病例达到2 197例；阿根廷登革热确诊病例为37.9万余例。1978年，我国首次在广东省佛山市发现了登革热病例。至今，我国在广东、广西、海南、云南、浙江、福建、台湾、澳门和香港等地区报告了登革热的暴发或流行以及本地感染病例[1-3]；据统计，登革热病例高达655 324例，其中死亡病例为610例[3]。登革热是一种通常症状轻微或无症状的疾病，大多数患者会在1-2周内自愈。然而，在极少数情况下，登革热可能会导致严重并发症，甚至死亡[4]。登革热的典型症状包括高烧、剧烈头痛、眼球后疼痛、肌肉和关节痛、恶心、皮疹以及身体肿胀[5]。这种疾病主要通过蚊子叮咬传播，涉及的蚊种包括白纹伊蚊(Aedes albopictus)和埃及伊蚊(Aedes aegypti)。目前，尚无特定药物可以治疗登革热，因此预防和控制疾病的传播显得尤为重要。
目前，登革热的预防主要集中在消灭媒介蚊子上，采用的方法包括物理方法和化学方法。化学方法涉及使用不同类型的杀虫剂，如菊酯类杀虫剂、有机磷类杀虫剂以及四氟苯菊酯定量气雾剂[6-8]。然而，化学杀虫剂的使用不仅会对环境造成污染，还可能对非目标物种造成伤害。此外随着时间的推移，蚊子可能会产生抗药性。RNA干扰(RNA interference, RNAi)是自然界生物独有的一种特异性抵御外来病毒和微生物入侵的保护机制，同时也是一种基因调控机制，参与机体的生长、代谢和发育调控[9]。RNAi的作用实现依赖于双链RNA (double-stranded RNA, dsRNA)的传递方法，从而使其开发过程更便捷[10]。由于RNAi容易被无处不在的细菌酶降解，因此它不会对环境造成危害[11-12]。如今，RNAi技术已被应用于蚊虫控制领域。Mysore等[13]设计了酿酒酵母的shRNA结构，并将其制作成酵母片饲喂蚊子幼虫，吞食重组酵母的蚊子幼虫靶基因得到有效沉默，最终导致蚊虫死亡。蚊子幼虫生活在水中，以小球藻、微藻、细菌等为食。费小雯等[14]针对蚊子的特性设计了V-ATPA基因的RNAi结构，并将其应用于莱茵衣藻中；通过使用重组微藻饲喂伊蚊幼虫，观察到幼虫的死亡率达到100.0%，并且与对照组相比，相关基因的表达量下降了97.4%。Carpenter等[15]通过RNAi技术成功使tor target of rapamycin (Tor)激酶基因的表达沉默，结果导致了卵黄原蛋白表达量的降低，并直接抑制了雌蚊卵子的产生。由此可见，RNAi技术在蚊虫防控方面的应用已经在理论和实验室层面取得了良好进展，为未来在实际环境中管理蚊虫种群提供了重要的指导意义。
γ-氨基丁酸(γ‑aminobutyric acid, GABA)是一种重要的抑制性神经递质，它可以通过转运蛋白(GABA transporter, GAT)进入神经元或神经胶质细胞中。GAT属于Na+/Cl-转运蛋白家族(SLC6基因家族)，在维持胞外GABA浓度稳定和突触间隙信号终止方面起着重要作用[16-17]。据报道，对于昆虫而言，GABA可以调节多种生理行为，如交配持久性[18]、摄食限制[19]、运动[20]、睡眠[21]、生物钟[22]、对乙醇的反应[23]和嗅觉记忆[24]。GAT在生理和病理状态下主要通过调控细胞外的GABA水平来发挥作用。当gat基因的表达出现异常时，会导致GABA介导的神经信号传递发生变化，这种变化会影响到GABA能神经元的活动，从而在维持神经系统的兴奋性与抑制性平衡中扮演核心角色。为此，本研究选择突触信号传递通路中的gat基因作为新的生物杀蚊靶点，将其转入莱茵衣藻(Chlamydomonas reinhardtii) CC124和小球藻(Chlorella vulgaris) HOC5中。将重组藻株饲喂白纹伊蚊幼虫和成虫后，重组藻株能在伊蚊体内释放针对gat基因的shRNA，特异性地沉默伊蚊体内的gat基因，从而使其无法转运GABA蛋白，阻断伊蚊的正常生态发育并导致其死亡，这可为蚊媒传染病的防控提供新思路。
1 材料与方法
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1.1 材料
白纹伊蚊捕捉于中国热带农业科学院院内，由本实验室继代保存。莱茵衣藻CC124购自中国科学院淡水藻种库(https://algae.ihb.ac.cn/)；小球藻HOC5为海南本地物种，分离纯化后接于Tris-acetate-phosphate (TAP)固体培养基保存。柱式质粒DNA小量抽提试剂盒购自Omega公司；植物DNA提取试剂盒、RNA提取试剂盒均购自广州美基生物科技有限公司；潮霉素B购自北京索莱宝科技有限公司；反转录试剂盒购自北京康为世纪生物科技有限公司；AceQ® qPCR SYBR Green Master Mix (Low ROX Premixed)购自南京诺唯赞生物科技股份有限公司；限制性内切酶Bgl Ⅱ、Sph Ⅰ、BstE Ⅱ均购自宝生物工程(大连)有限公司。pCAMBIA1302载体由深圳华大基因科技有限公司惠赠。
1.2 白纹伊蚊 gat 基因shRNA靶位点选择与设计
根据NCBI上已公布的伊蚊gat基因的mRNA序列(GenBank登录号为XM_019696122)，通过线上设计网站GPP (http：//www.broadinstitute.org/rnai/public/seq/search)筛选出分数最高的几条shRNA靶序列，并进行BLAST比对，排除与其他非相关基因同源的序列后，选择合适的作为靶序列。shRNA模板中选用CTCGAG序列作为中间loop环结构。根据pCAMBIA1302载体的酶切位点要求，在正义链的5′端添加GATC，可与Bgl Ⅱ酶切pCAMBIA1302载体后的黏性末端互补；在反义链的5′端添加GTCACC，可与BstE Ⅱ酶切pCAMBIA1302载体后的黏性末端互补。设计好的shRNA序列包含酶切位点、正义链、loop环、反义链、终止信号和酶切位点，其序列见表1。
1.3 shRNA RNAi表达载体构建及鉴定
1.3.1 目的单链片段退火成双链
将设计合成的正、反向寡核苷酸单链(100 μmol/L)各1 µL加入48 μL的10×H buffer (稀释10倍)反应体系中(引物终浓度 2 μmol/L)，从95 ℃缓慢退火至室温，形成两端分别具有Bgl Ⅱ和BstE Ⅱ黏性末端的寡核苷酸双链。
1.3.2 pCMBIA1302载体的限制性酶切
先后使用Bgl Ⅱ和BstE II限制酶对pCAMBIA1302载体进行双酶切，通过1%琼脂糖凝胶电泳鉴定并用柱式DNA胶回收试剂盒回收。
1.3.3 连接与转化
将酶切后回收的载体与退火产物在16 ℃下连接过夜(T4 DNA连接酶)。连接产物转入TOP10感受态细菌中，并涂布于含有卡那霉素(100 μg/mL)的LB平板上，37 ℃恒温培养过夜(12-16 h)。第2天，挑取阳性单菌落于50 mL含有卡那霉素的LB液体培养基中，37 ℃、180 r/min培养10-12 h，提取质粒，并用Sph I酶切鉴定重组质粒。未连接干扰片段的空载体pCAMBIA1302质粒作为对照。将构建好的重组质粒命名为gat-p1302。
1.4 shRNA RNAi重组微藻的转化、筛选与鉴定
1.4.1 电击法转化莱茵衣藻、小球藻
采用Wang等[25]的实验参数，对电击条件进行调整。调整后的条件：电压500 V；脉冲长度4 MS；脉冲次数7次；间隔时间100 ms。
1.4.2 筛选后阳性藻株的DNA提取及PCR鉴定
使用植物DNA提取试剂盒提取工程藻株的DNA，并进行PCR反应。引物序列见表1中的pCAMBIA1302引物。PCR反应体系(25 μL)：2×Flash PCR MasterMix 12.5 μL，上、下游引物(10 µmol/L)各1 μL，DNA模板1 µL，ddH2O 9.5 µL。PCR反应条件：98 ℃预变性30 s；94 ℃变性10 s，60 ℃退火15 s，72 ℃延伸15 s，共35个循环；72 ℃终延伸1 min。通过2%琼脂糖凝胶电泳鉴定gat-p1302表达载体是否成功转入微藻中。
1.4.3 伊蚊的饲养
蚊虫的孵育条件：温度27-29 ℃，强光照射，添加饲料，直至成蚊羽化。成蚊生长条件：温度27 ℃，相对湿度70%，使用10%蔗糖溶液饲喂成蚊，每48 h提供1次小白鼠供雌蚊吸血。
1.4.4 伊蚊幼虫的生物学检测
将阳性工程藻株用于饲喂伊蚊幼虫，每组30只幼虫，重复3次。阳性工程藻饲喂伊蚊幼虫4 d后，每组随机选取5只幼虫测量体长。
饲喂不同转基因藻株4 d后，使用RNA提取试剂盒提取伊蚊幼虫总RNA，使用反转录试剂盒进行反转录，得到cDNA后，以白纹伊蚊体内核糖体蛋白S17 (ribosomal protein S17，Gene ID：109416926)作为内参基因进行qPCR检测。引物序列见表1。qPCR反应体系(20 µL)：2×AceQ® qPCR SYBR Green Master Mix 10 µL，上、下游引物(10 µmol/L)各0.4 µL，cDNA模板1 µL，ddH2O 8.2 µL。反应条件：95 ℃ 5 min； 95 ℃ 10 s，60 ℃ 30 s，共40个循环； 95 ℃ 15 s，60 ℃ 60 s；95 ℃ 15 s。
1.4.5 糖毒诱饵试验
野生型CC124藻株与10%蔗糖溶液混合作为阴性对照；0.1 mg/L溴氰菊酯与10%蔗糖溶液混合作为阳性对照；重组藻株与10%蔗糖溶液混合作为实验组。上述溶液按1:1比例混合后用于饲喂伊蚊成蚊。每组50只成蚊，重复3次，实验为期7 d，每天统计蚊虫死亡情况。
2 结果与分析
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2.1 shRNA RNAi表达载体的验证
使用限制性内切酶Sph I对构建好的p1302-gat重组质粒进行单酶切，以未连接干扰片段的空载pCAMBIA1302质粒作为对照。如图1B所示，pCAMBIA1302空载质粒大小为10 549 bp，经Sph I酶切后得到2条片段，大片段大小为8 619 bp，小片段为1 930 bp，重组质粒酶切后，大片段大小为8 619 bp，小片段为1 234 bp，与预期结果一致，说明载体构建成功。
2.2 阳性shRNA RNAi重组微藻的获得
将准备好的质粒与生长至对数期的CC124和HOC5分别进行电击转化。分别以ddH2O组转化CC124和HOC5作为阴性对照，以空载体pCAMBIA1302转化微藻作为阳性对照。电击转化后，将藻株涂布在含有25 μg/mL和150 μg/mL潮霉素B的TAP固体培养基上，筛选阳性工程藻株。将筛选后的单个藻株挑入50 mL TAP液体培养基中培养至对数期(OD值为0.3-0.5)，提取微藻基因组DNA进行 PCR鉴定。2%琼脂糖凝胶电泳部分结果见图1C，阳性藻株扩增的目的片段大小约为475 bp。结果显示，成功获得转gat-shRNA小球藻5株、转gat-shRNA莱茵衣藻6株，以及转空载体pCAMBIA1302的转基因藻株5株。
2.3 饲喂shRNA重组微藻对伊蚊幼虫进行RNA干扰
2.3.1 饲喂shRNA重组微藻对伊蚊幼虫的影响
为了探究gat基因沉默对伊蚊幼虫的致死效果，将成功转化的RNAi重组微藻用于饲喂伊蚊幼虫，设置2组实验。第1组：对照组(饲料组、CC124组、pCAMBIA1302组)和实验组(转莱茵衣藻gat 1-5号转化子)；第2组：对照组(饲料组、HOC5组、pCAMBIA1302组)和实验组(转小球藻gat 1-5号转化子)，每组重复3次。观察发现，饲喂重组莱茵衣藻和小球藻的伊蚊幼虫生长发育迟缓，身体出现轻微颤抖，对光照反应迟钝。幼虫死亡率见图2，饲喂重组CC124藻株的伊蚊幼虫在饲喂第2天开始出现死亡现象，观察15 d后，饲喂野生CC124藻株的幼虫死亡率为27.78%，饲喂空载体p1302藻株的幼虫死亡率为30.00%，而饲喂转莱茵衣藻gat基因的幼虫死亡率最高，达到75.56%；饲喂重组HOC5藻株的伊蚊幼虫在第1天开始出现死亡，第2天至第10天死亡速度较快，第10天后死亡速度逐渐减缓，饲喂野生HOC5藻株的幼虫死亡率为0，饲喂转空载体p1302藻株的幼虫死亡率为2.22%，而饲喂转小球藻gat基因的幼虫死亡率为62.22%。通过SPSS软件(IBM SPSS Statistics 19)进行单因素方差分析，结果显示在饲喂莱茵衣藻组中处理组1-5号转化子与对照组之间白纹伊蚊的死亡率差异显著(P<0.05)，但各重组藻株转化子之间的差异性不显著(P>0.05)；在饲喂小球藻组中，处理组1-5号转化子与对照组之间白纹伊蚊的死亡差异显著(P<0.05)，但各重组藻株转化子之间死亡差异不显著(P>0.05)，说明饲喂gat-shRNA重组莱茵衣藻和小球藻对伊蚊幼虫具有显著的致死效果。
2.3.2 饲喂shRNA重组微藻对伊蚊幼虫体长的影响
根据2.3.1节的结果，重组微藻对伊蚊幼虫的生长发育有显著影响。因此，设置2组实验，饲喂伊蚊幼虫4 d后，每组随机挑取5只幼虫，在显微镜下测量其体长(图3、图4)。饲喂清水组、饲料组和野生CC124藻株组的幼虫平均体长分别为1 888.40、5 865.00和3 974.20 μm；饲喂转莱茵衣藻gat 1-5转化子组的幼虫平均体长分别为3 086.40、3 040.40、3 079.20、3 451.40和3 215.80 μm；饲喂野生小球藻藻株组的幼虫平均体长为4 503.00 μm，饲喂转小球藻gat 1-5转化子组的幼虫平均体长分别为2 851.33、2 860.67、2 634.33、2 658.33和2 513.67 μm，说明实验组幼虫的体长明显短于对照组(P<0.05)，表明gat基因的沉默能够延缓伊蚊幼虫的发育。
2.3.3 饲喂shRNA微藻对重组伊蚊幼虫靶基因表达的影响
为了检测伊蚊幼虫饲喂重组微藻是否有效沉默靶基因的表达，选取饲喂第4天的幼虫通过qPCR技术检测靶基因表达水平。以饲喂野生CC124藻株、野生HOC5藻株和转空载体pCAMBIA1302的微藻为对照组，以RpS17作为内参基因。测定结果见图5，饲喂gat-shRNA莱茵衣藻的幼虫靶基因相对表达量为55.93%，饲喂gat-shRNA小球藻的幼虫靶基因相对表达量为20.46%，而对照组的相对基因表达量为100.00%。结果表明，饲喂重组微藻的伊蚊幼虫靶基因表达显著下调，说明该RNAi重组微藻能够有效沉默靶基因，且处理组与对照组之间的差异显著(P<0.05)。
2.4 饲喂shRNA重组微藻对伊蚊成虫致死情况
本研究分为3组：野生CC124藻株+10%蔗糖作为阴性对照；溴氰菊酯+10%蔗糖作为阳性对照；gat-shRNA莱茵衣藻+10%蔗糖作为实验组。成蚊落在蚊笼底端且无活动能力视为死亡。观察7 d的成蚊致死结果见图6，饲喂无毒野生莱茵衣藻CC124混合液的成蚊最终死亡率为20.67%，饲喂有毒的溴氰菊酯混合液的成蚊在第5天死亡率即达100.00%，而饲喂有毒的gat-shRNA莱茵衣藻混合液的成蚊死亡率为58.67%。与无毒CC124组相比，实验组对伊蚊成虫具有显著的致死效果。
3 讨论与结论
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RNAi技术通过降低特定基因的表达来控制昆虫种群，为昆虫治理提供了一种新的方案[26-27]。特别是在蚊子研究领域，RNAi不仅被用来探究蚊子的基因功能，还用于研究蚊子与病原体之间的相互作用[28]。在运用RNAi技术进行蚊子防控方面，目前主要采用的方法包括注射、浸泡、利用纳米颗粒以及通过微生物将双链RNA (dsRNA)传递给蚊子幼虫[28-30]。例如，费小雯等[14]针对蚊子的特性设计了V-ATPA基因的RNAi结构，并将此技术应用于莱茵衣藻中；通过使用重组微藻饲喂伊蚊幼虫，观察到幼虫的死亡率达到了100%。此外，费小雯等[31]构建了针对埃及伊蚊3HKT基因的RNAi结构，并将其转化到莱茵衣藻中，用其饲喂蚊子幼虫，结果表明，这种方法对伊蚊幼虫也具有100%的致死率。相比之下，本研究中使用的转基因藻类对白纹伊蚊幼虫的致死率为62.22%-75.56%。致死效果的差异可能源于目标物种的不同以及所选择的RNAi沉默片段的差异。利用微藻作为介导RNA干扰(RNAi)的技术，可以高效地实现目标基因的敲除，这种方法不仅成本低廉、环保，而且不会增加蚊虫的抗药性。使用莱茵衣藻和小球藻作为外源RNAi的载体，通过让幼虫和成虫摄取经过RNAi重组的微藻来实现靶基因的敲除。本研究合成的RNAi外源片段是小发夹RNA (shRNA)，生成的小发夹RNA结构单一，专一性更强，且与RNA诱导沉默复合体的结合效率更高，能有效降低脱靶效应。
Gat基因编码的蛋白对神经递质的转运至关重要。通过饲喂幼虫和成虫RNAi重组微藻来研究gat基因在伊蚊中的功能，结果显示，当伊蚊的gat基因受到干扰时，与对照组相比伊蚊的死亡率显著增加(图2)，幼虫的生长发育也受到抑制，体长相对较短(图3、图4)，数据表明gat基因在伊蚊的生长发育中起到了关键作用。qPCR分析进一步证实，通过饲喂RNAi重组微藻可以显著降低靶基因的mRNA水平，从而成功地对gat基因进行了RNA干扰(图5)。糖毒诱饵实验是一种相对较新发展的诱捕技术，具有高特异性、可持续和低成本等特点[32-33]。在糖毒诱饵实验中引入重组微藻gat-shRNA后，24 h观察到显著的致死效果，表明在幼虫期间储备的能量已基本耗尽，成蚊渴望寻找更多食物来维持生存。尽管阴性对照组也有死亡，但处理组的死亡率为58.67%，高于阴性对照(图6)，而阳性对照组的死亡率为100.00%。虽然处理组的死亡率略低于溴氰菊酯阳性糖毒诱饵的致死率，但使用重组微藻比使用溴氰菊酯杀虫剂更安全、更环保。
综上所述，使用gat RNAi重组微藻对蚊子进行处理显示出了明显的杀虫效果。由于微藻可以作为伊蚊幼虫的食物，并且在自然界中广泛存在，因此gat RNAi重组微藻在商业应用方面具有巨大潜力。然而，白纹伊蚊的生理过程极为复杂，目前的研究仅局限于mRNA表达水平的变化，对于蛋白质层面的影响尚未深入探讨。因此，未来的研究需要进一步扩展至蛋白质层面，以全面理解gat基因干扰的作用机制。同时，基于微藻RNAi的技术为蚊虫种群控制提供了新的途径。
基金
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  • 海南省重点研发计划(ZDYF2022SHFZ314)
  • 国家自然科学基金(82260669)
  • 海南省自然科学基金(322RC766)
  • 农业农村部财政专项(NFZX2024)
文献
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文章信息
doi: 10.13343/j.cnki.wsxb.20240781
  • 接收时间：2024-12-04
  • 首发时间：2026-02-05
  • 出版时间：2025-05-04
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  • 收稿日期：2024-12-04
  • 录用日期：2024-12-31
基金
Key Research and Development Program of Hainan Province(ZDYF2022SHFZ314)
海南省重点研发计划(ZDYF2022SHFZ314)
National Natural Science Foundation of China(82260669)
国家自然科学基金(82260669)
Natural Science Foundation of Hainan Province(322RC766)
海南省自然科学基金(322RC766)
Financial Special Project of the Ministry of Agriculture and Rural Affairs(NFZX2024)
农业农村部财政专项(NFZX2024)
作者信息
    1.佳木斯大学 生物与农业学院，黑龙江 佳木斯
    2.中国热带农业科学院热带生物技术研究所，海南省海洋生物资源功能性成分研究与利用重点实验室，海南 海口
    3.海南医科大学 基础医学与生命科学学院，海南 海口
    4.中国热带农业科学院湛江实验站，广东 湛江
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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
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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