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舞毒蛾LdOR2基因克隆及对CO2胁迫下的行为反应

王建国 王建军 曹传旺 孙丽丽

王建国, 王建军, 曹传旺, 孙丽丽. 舞毒蛾LdOR2基因克隆及对CO2胁迫下的行为反应[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210184
引用本文: 王建国, 王建军, 曹传旺, 孙丽丽. 舞毒蛾LdOR2基因克隆及对CO2胁迫下的行为反应[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210184
Wang Jianguo, Wang Jianjun, Cao Chuanwang, Sun Lili. Cloning of LdOR2 gene in Lymantria dispar and its behavioral response to CO2 stress[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210184
Citation: Wang Jianguo, Wang Jianjun, Cao Chuanwang, Sun Lili. Cloning of LdOR2 gene in Lymantria dispar and its behavioral response to CO2 stress[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210184

舞毒蛾LdOR2基因克隆及对CO2胁迫下的行为反应

doi: 10.12171/j.1000-1522.20210184
基金项目: “十三五”国家重点研发计划(2018YFC1200400)
详细信息
    作者简介:

    王建国。主要研究方向:昆虫生理生化与分子毒理学。Email:1977756072@qq.com 地址:150040 黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林学院

    责任作者:

    孙丽丽,副教授。主要研究方向:昆虫生理生化与分子毒理学。Email:sunlilinefu@126.com 地址:同上

Cloning of LdOR2 gene in Lymantria dispar and its behavioral response to CO2 stress

  • 摘要:   目的  本文克隆了舞毒蛾的气味受体基因LdOR2,并阐明该基因在舞毒蛾各发育期和组织中的表达特征及其对CO2浓度胁迫下的行为响应,为进一步研究气候变化下舞毒蛾的嗅觉反应机制提供理论依据。  方法  通过转录组文库筛选克隆出LdOR2基因,利用生物信息学分析其基因特性,通过实时荧光定量PCR(RT-qPCR)技术检测LdOR2基因在不同发育阶段和组织以及不同CO2浓度(397、550和750 μL/L)下的表达水平,并利用RNA干扰(RNAi)技术研究不同CO2浓度下LdOR2基因沉默后舞毒蛾的行为学反应。  结果  舞毒蛾LdOR2基因开放阅读框(ORF)为1 203 bp,编码400个氨基酸,蛋白分子量为45.76 kDa,理论等电点为8.22;进化树分析结果表明,舞毒蛾LdOR2与黏虫MsepOR24和双委夜蛾AdisOR21亲缘关系较近,并聚为一类;RT-qPCR结果显示,LdOR2在舞毒蛾各发育阶段均有表达,在雌蛹中表达量最高,雄成虫中表达量最低;在雌、雄成虫不同组织中,雌、雄触角中表达量显著高于其它组织(P < 0.05),但雌、雄虫触角间的表达量差异不明显。高CO2浓度下LdOR2基因表达量降低,其中,550 μL/L和750 μL/L条件下雌虫触角中其表达量与对照组相比分别下降21%和29%(P < 0.05),雄虫触角中其表达量与对照组相比分别下降了43%和7%(P < 0.05)。LdOR2基因沉默后,舞毒蛾雌、雄成虫对丁香酚和顺-3-己烯-1-醇的趋向性减弱,而在高浓度CO2处理条件下,舞毒蛾沉默体对7种挥发物的反应率均有所下降。  结论  舞毒蛾LdOR2在其气味识别过程中发挥重要作用,CO2浓度变化通过调节舞毒蛾LdOR2基因的表达进而影响其对气味的敏感性。

     

  • 图  1  14种昆虫OR蛋白系统进化树

    Figure  1.  Phylogenetic analysis of OR proteins from 14 insects

    图  2  舞毒蛾发育阶段LdOR2基因表达量

    E表示卵期;1L ~ 6L表示1 ~ 6龄幼虫;P(F)和P(M)分别表示雌蛹和雄蛹;A(F)和A(F)分别表示雌成虫和雄成虫。不同小写字母表示发育的不同阶段基因表达差异显著性(P < 0.05)。下同。Note: E indicate egg stage; 1L ~ 6L indicate 1st to 6th instar larvae; P(F) and P(M) indicate female and male pupae; A(F) and A(M) indicate female and male adult. Different lowercase letters indicate significant differences in gene expression at different developmental stages of development (P < 0.05). The same below.

    Figure  2.  Expression level of LdOR2 gene among different developmental stages in L. dispar

    图  3  舞毒蛾不同成虫组织LdOR2基因表达量

    A. 雌成虫;B. 雄成虫。Note: A, female adult; B, male adult.

    Figure  3.  Expression level of LdOR2 gene among different developmental tissues in L. dispar adults

    图  4  不同CO2浓度下舞毒蛾触角LdOR2基因表达量

    Figure  4.  Expression levels of LdOR2 in L. dispar antenna under different CO2 concentrations

    图  5  不同CO2浓度处理下舞毒蛾LdOR2基因沉默效率

    A. 397 μL/LCO2浓度下沉默效率;B. 550 μL/LCO2浓度下沉默效率;C. 750 μL/LCO2浓度下沉默效率;星号表示处理组dsOR2和对照组dsRed基因表达量显著性差异(** P < 0.01;*** P < 0.001;t检验)。Note: A, Silencing efficiency at 397 μL/LCO2 concentrations; B, Silencing efficiency at 550 μL/LCO2 concentrations; C, Silencing efficiency at 750 μL/LCO2 concentrations; Asterisk indicates significant difference in gene expression between treatment group and control group (** P < 0.01; *** P < 0.001; t-test).

    Figure  5.  Silencing efficiency of LdOR2 gene in L. dispar larvae under different CO2 concentrations

    图  6  不同CO2浓度舞毒蛾LdOR2基因沉默体雌成虫行为反应

    A. 丁香酚;B. 石竹烯;C. 水杨醛;D. 顺3-己烯-1-醇;E. α-蒎烯;F. 苯甲醛;G. 邻苯二甲酸二异丁酯;1. 397 μL/LCO2浓度注射dsRed;2. 397 μL/LCO2浓度注射dsOR2;3. 550 μL/LCO2浓度注射dsRed;4. 550 μL/LCO2浓度注射dsOR2;5. 750 μL/LCO2浓度注射dsRed;6. 750 μL/LCO2浓度注射dsOR2;星号表示对照组(石蜡)与处理组挥发物差异显著(* P < 0.05;** P < 0.01;t检验)。下同。Note: A: Eugenol; B: Caryophyllene; C: Salicylaldehyde; D: cis-3-Hexen-1-ol; E: α-Pinene; F: Benzaldehyde; G: Dibutyl phthalate; 1: Injection dsRed at a concentration of 397 μL/LCO2; 2: Injection dsOR2 at a concentration of 397 μL/LCO2; 3: Injection dsRed at a concentration of 550 μL/LCO2; 4: Injection dsOR2 at a concentration of 550 μL/LCO2; 5: Injection dsRed at a concentration of 750 μL/LCO2; 2: Injection dsOR2 at a concentration of 750 μL/LCO2; Asterisk indicates a significant difference in volatiles between the contral group (paraffin) and the treatment group (* P < 0.05; ** P < 0.01; t-test). The same below

    Figure  6.  Behavioral responses of female L. dispar adults with LdOR2 gene silencing under different CO2 concentrations

    图  7  不同CO2浓度下舞毒蛾LdOR2基因沉默体雄虫行为学反应

    Figure  7.  Behavioral responses of male L. dispar adults with LdOR2 gene silencing under different CO2 concentrations

    表  1  舞毒蛾LdOR2基因特性分析生物信息学软件

    Table  1.   Bioinformatics software for gene characteristics analysis of LdOR2 in L. dispar

    项目 Item软件 Software
    开放读码框 Open reading frame ORF finder http://www.ncbi.nlm.nih.gov/gorf.html
    分子量及理论等电点 Molecular weight and theoretical isoelectric point ProtParam http://au.expasy.org/tools/protparam.html
    信号肽序列检测 Signal peptide sequence detection SignlP4.1 Server http://www.cbs.dtu.dk/services/SignalP
    保守区预测 Conservative prediction http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
    同源性分析 Homology analysis Blast http://www.ncbi.nlm.nih.gov/BLAST/
    多序列比对 Multiple sequence alignment Clustalx(1.83)
    系统发育树构建 Phylogenetic tree construction MEGA(5.1)
    下载: 导出CSV

    表  2  本文所用引物序列

    Table  2.   Primer sequences used in this study

    基因
    Gene
    正向引物序列(5′-3′)
    Forward primer sequence (5′-3′)
    反向引物序列(5′-3′)
    Reverse primer sequence (5′-3′)
    序列片段大小
    Sequence fragment length/bp
    引物用途
    Primer usage
    dsOR2 TAATACGACTCACTATAGGGAG
    GCGATGATCGAAACTTGC
    TAATACGACTCACTATAGGGCG
    GACACAATCATAGTCACCA
    534 dsRNA合成
    dsRNA synthesis
    dsRed TAATACGACTCACTATAGGG
    GAGAACGTCATCACCGAGTT
    TAATACGACTCACTATAGGG
    GATGGTGTAGTCCTCGTTGT
    658
    LdOR2 GAGTTTCGCCGTCAGTCACA CACGCGCATGAACCGTAAAC 201 实时荧光定量
    Real time fluorescent quantitation
    Actin AGAAGCACTTGCGGTGGACAAT ACCTGTACGCCAACACTGTCAT 252
    TUB AATGCAAGAAAGCCTTGCGCCT ATGAAGGAGGTCGACGAGCAAA 235
    EF1α TTTGCCTTCCTTGCGCTCAACA TGTAAAGCAGCTGATCGTGGGT 223
    注:dsOR2是LdOR2基因的双链RNA;dsRed是对照基因的双链RNA。Note: dsOR2 is double-stranded RNA of the LdOR2; dsRed is double-stranded RNA of the control gene.
    下载: 导出CSV

    表  3  试验中所使用的气味源

    Table  3.   The list of odors used in the experimentss

    化合物名称
    Compound name
    CAS登录号
    CAS registry number
    纯度
    Purity/%
    丁香酚 Eugenol 97-53-0 99.0
    水杨醛 Salicylaldehyde 90-02-8 99.0
    苯甲醛 Benzaldehyde 100-52-7 98.0
    反式石竹烯 trans-Caryophyllene 87-44-5 80.0
    邻苯二甲酸二异丁酯 Dibutyl phthalate 84-66-2 99.0
    顺-3-己烯-1-醇 cis-3-Hexen-1-ol 928-96-1 98.0
    α-蒎烯 α-Pinene 80-56-8 98.0
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-05-14
  • 修回日期:  2021-06-07
  • 网络出版日期:  2021-08-03

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