Cloning of LdOR2 gene in Lymantria dispar and its behavioral response to CO2 stress
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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。Abstract:Objective In this study, we cloned the odorant receptor gene (LdOR2) and determined the expression levels of this gene in developmental stages and different tissues of the Lymantria dispar and its behavioral response to CO2 stress. The results will provide a theoretical basis for clarifying the olfactory response mechanism of the L. dispar under climate change.Method The LdOR2 gene was cloned through transcriptome library screening, and its characteristics were analyzed by bioinformatics. The expression levels of LdOR2 gene in different developmental stages and tissues as well as in different CO2 concentrations (397, 550 and 750 μL/L) were determined by real-time fluorescence quantitative PCR technology. In addition, RNA interference (RNAi) technology was used to study the behavioral responses of L. dispar adults silenced by LdOR2 at different CO2 concentrations.Result The open reading frame (ORF) of LdOR2 gene in L. dispar was 1 203 bp, encoding 400 amino acids. The molecular mass of the LdOR2 protein was 45.76 kDa and the theoretical isoelectric point was 8.22. The phylogenetic tree showed that the LdOR2 in L. dispar was closely related to MsepOR24 Mythimna separata and AdisOR21 in Athetis dissimilis, and clustered into one group. RT-qPCR results showed that LdOR2 was expressed at all developmental stages of the L. dispar, with the highest expression level in female pupae and the lowest expression levels in male adults. In different tissues of female and male adults, the expression levels in antennae were significantly higher than those in other tissues (P < 0.05), but showed no difference between the antennae of female and male. The expression of LdOR2 gene decreased under high CO2 concentration. Compared with the control group, the expression of female antennae under 550 μL/L and 750 μL/L conditions decreased by 21% and 29% (P < 0.05), respectively, and the expression levels of antennae of male L. dispar adults decreased by 43% and 7% (P < 0.05). After LdOR2 gene silencing, the tendency of female and male L. dispar adults to eugenol and cis-3-hexene-1-ol was weakened, while the response rates of the L. dispar silencers to seven volatiles decreased under high CO2 concentration.Conclusion LdOR2 plays an important role in the odor recognition of L. dispar. The sensitivity of L. dispar to odor was affected by the expression levels of LdOR2 gene regulated by the changes of CO2 concentration.
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Keywords:
- Lymantria dispar /
- LdOR2 /
- cloning and expression /
- CO2 concentration /
- odorant receptor /
- RNA interference /
- behavioral response
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全球气候变暖已经引起世界范围内的关注,据IPCC报道,CO2浓度在2005年间达到397 μL/L,并预测在21世纪中叶将达到约550 μL/L,21世纪末将达到约800 μL/L[1]。大气CO2浓度的不断升高会直接或间接影响森林生态系统,同时昆虫对CO2的浓度变化也极其敏感[2]。昆虫的嗅觉识别过程需要多种蛋白的共同参与,包括气味结合蛋白(odorant binding proteins,OBPs)、化学感受蛋白(chemosensory proteins,CSPs)、气味受体(odorant receptors,ORs)、离子受体(inotropic receptors,IRs)、感觉神经元膜蛋白(sensory neuron membrane proteins,SNMPs)和气味降解酶(odor degrading enzymes,ODEs)[3]。昆虫的气味受体能够识别气味分子,并能将小分子化学信号转变为电信号[4]。在昆虫对外界气味识别的过程中,气味受体对气味分子的专一性识别,使其成为嗅觉系统的关键[5]。气味受体分为两类:一种是非典型受体(odorant receptor co-receptor,Orco),在物种之间高度保守;另一种是传统气味受体(conventional odorant receptors,ORx),在物种间高度分化[6]。继首次在黑腹果蝇(Drosophila melanogaster)中成功克隆出第一个气味受体OR基因后[7],目前已经在不同种类的昆虫中鉴定出了大量的OR基因,例如:冈比亚按蚊(Anopheles gambiae)中鉴定到79个ORs基因[8],家蚕(Bombyx mori)中有64个ORs基因[9]。
舞毒蛾(Lymantria dispar)是一种世界性林业食叶害虫,能够危害多种寄主植物[10]。嗅觉系统在昆虫与外界化学信息交流的过程中起到了关键的作用,因此,对舞毒蛾嗅觉系统的深入研究是防治其危害的一个突破点。在气候变暖的前提下研究CO2浓度升高对昆虫嗅觉系统的响应,更具有现实意义。目前舞毒蛾嗅觉相关基因的研究主要集中在OBPs、CSPs等基因的克隆及对这些基因的表达分析[11],而对于普通气味受体(ORs)的研究比较少。前期我们在舞毒蛾转录组文库中筛选获得4条OR基因(OR1 ~ OR4),并测定了CO2胁迫对4个OR基因表达的影响,结果发现LdOR2基因随CO2浓度(397、550和750 μL/L)的升高,转录水平显著抑制下调(未发表)。本文在此基础上克隆获得LdOR2基因,进行基因序列分析及表达分析。通过RNA干扰(RNAi)技术探索不同CO2浓度对舞毒蛾LdOR2基因表达的影响,为探讨高浓度CO2下舞毒蛾种群适应性提供理论依据。
1. 材料与方法
1.1 供试昆虫与处理
舞毒蛾卵和人工饲料购于中国林业科学研究院森林生态环境与保护研究所,卵在10%甲醛溶液中浸泡1 h,用清水反复冲洗干净后放入光周期14L∶10D、温度(25 ± 1)℃、湿度(70 ± 5)%的CO2人工气候培养箱中饲养。CO2浓度分别设置为550和750 μL/L,对照组为正常大气CO2浓度(397 μL/L)。
1.2 LdOR2基因克隆与分析
采用RNeasy Mini动物组织总RNA提取试剂盒,提取各龄期总RNA,由深圳华大基因科技有限公司进行转录组文库构建及测序。获得的Unigenes进行Blastx和Blastn分析,根据功能注释结果选择具有完整ORF的OR基因,设计引物用于RT-qPCR验证,测序验证获得LdOR2基因序列。利用表1中软件及程序对LdOR2进行生物信息学分析。
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 mass 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) 1.3 实时荧光定量RT-qPCR
提取舞毒蛾各发育阶段(卵、幼虫、雄蛹、雌蛹、雄成虫、雌成虫)以及雌、雄成虫各组织(头、触角、胸、腹、足)RNA。用DNaseI(Promega)去除总RNA中的DNA,测定其质量浓度,取0.5 μg RNA用于cDNA合成,按照PimeScriptTM RT reagent Kit(Takara)合成cDNA。反应条件:42 ℃ 60 min,85 ℃ 5 s,16 ℃ 10 min。将cDNA稀释10倍备用。内参基因(Actin、TUB和EF1α)和LdOR2基因的引物序列见表2。反应体系为:2 × SYBR premix Ex Taq酶10 μL,上、下游引物(10 μmol/L)各1 μL,稀释后的cDNA 2 μL,去离子水补足至20 μL。反应条件:94 ℃ 30 s;94 ℃ 12 s,58 ℃ 45 s,72 ℃ 40 s,81 ℃ 1 s,循环数为45,每个样品重复3次,用2−ΔΔCt方法进行基因相对表达水平分析[12]。
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 usagedsOR2 TAATACGACTCACTATAGGGAG
GCGATGATCGAAACTTGCTAATACGACTCACTATAGGGCG
GACACAATCATAGTCACCA534 dsRNA合成
dsRNA synthesisdsRed TAATACGACTCACTATAGGG
GAGAACGTCATCACCGAGTTTAATACGACTCACTATAGGG
GATGGTGTAGTCCTCGTTGT658 LdOR2 GAGTTTCGCCGTCAGTCACA CACGCGCATGAACCGTAAAC 201 实时荧光定量
Real time fluorescent quantitationActin 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。Notes: dsOR2 is double-stranded RNA of the LdOR2; dsRed is double-stranded RNA of the control gene. 1.4 RNA干扰
根据已获得的LdOR2基因序列,设计引物,引物序列见表2,PCR扩增目的基因片段。参照MEGAscript RNAi Kit试剂盒(Ambion)说明合成dsRNA,用于进行LdOR2基因沉默。电泳检测dsRNA完整性,将dsRNA稀释至1000 ng/μL,冻存于−80 ℃待用。
不同CO2浓度处理组(550和750 μL/L)和对照组(397 μL/L)舞毒蛾卵期饲养至蛹期,分别选取同一天化蛹的大小一致的舞毒蛾蛹,微量注射1.0 μg dsRNA至蛹的腹面,饲养至羽化。注射雌、雄蛹各40头,以dsRed为对照,每个处理重复3次。取注射后舞毒蛾触角进行总RNA提取,反转录成cDNA,用来检测RNAi后基因的沉默效率。
1.5 不同CO2浓度下干扰后的行为学反应
通过Y型嗅觉仪来测定舞毒蛾成虫对7种寄主植物杨树(Populus spp.)体内主要挥发物的行为反应[13-18]。试验中测试的化合物见表3,浓度为102 μg/μL(依据课题组前期舞毒蛾成虫电生理和Y型管试验筛选出的7种化合物最佳反应浓度,数据未发表),对照为液体石蜡。Y型嗅觉仪主臂长度为30 cm,而两侧臂长度为20 cm,玻璃管的内直径为7 cm。两侧臂分别连接样品瓶、加湿瓶及活性炭管,用微量移液器取10 μL样品滴于滤纸条上,将其放入其一样品瓶中,取滴有相同体积石蜡的滤纸条放入另一样品瓶内。打开大气采样仪持续进气30 s后释放舞毒蛾成虫,计时3 min,当试虫在某一侧臂超过5 cm,并持续在该侧臂停留超过1 min,则记为该试虫对位于该侧臂的气味源做出了选择。若该试虫在接入的3 min内不作出选择,则记为该试虫无反应,并结束试验。每一气味源测试3组,每组测试30头。每组测试5头后,调换两侧臂位置,每完成一组的测试后需要用丙酮和蒸馏水将管内壁冲洗干净,在烘箱中烘干备用。
化合物名称
Compound nameCAS登录号
CAS registry No.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 1.6 数据统计和分析
采用Excel2007计算表达量数据,运用SPSS17.0(SPSS Inc,USA)统计软件进行ANOVA分析,使用OriginPro8.5软件进行数据统计和绘图。
2. 结果与分析
2.1 LdOR2基因克隆和进化树分析
通过舞毒蛾转录组数据的分析和RT-qPCR验证获得LdOR2基因开放阅读框序列,LdOR2基因ORF长为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,为碱性蛋白,跨膜结构分析结果显示LdOR2蛋白具有6个跨膜螺旋结构,BLASTP对LdOR2蛋白保守区预测结果表明,该蛋白属于昆虫气味受体家族的7tm-6跨膜受体结构。
通过BLASTP多序列比对,选择与舞毒蛾OR序列相似程度高的14种昆虫的23个OR序列进行进化树分析(图1)。进化树分析结果表明:LdOR2与黏虫(Mythimna separata)QNS36221.1、浅灰色夜蛾(Athetis dissimilis)ALM26210.1亲缘关系较近并聚为一类。其中LdOR2与黏虫QNS36221.1同源性最高,为72.10%。
2.2 LdOR2基因发育和组织特异性
LdOR2在舞毒蛾各发育阶段均有表达(图2)。以卵期基因表达量为对照,LdOR2基因在舞毒蛾雌蛹期的表达量最高,为对照组的1.61倍,其余龄期均低于对照组;雄成虫表达量最低,仅为对照组的3.62%;在不同组织表达模式中,以头部的表达量为对照,LdOR2在舞毒蛾成虫各组织中均有表达,且均在触角中高表达。LdOR2基因在雌成虫触角中表达水平为头部的29.76倍(图3A);在雄成虫触角中LdOR2基因表达水平为头部的1.50倍(图3B)。
2.3 CO2浓度处理下舞毒蛾触角LdOR2表达
为了进一步明确CO2浓度变化对LdOR2基因表达水平的影响,采用RT-qPCR技术分析397 μL/L(大气浓度)、550 μL/L和750 μL/LCO2浓度下舞毒蛾触角中LdOR2基因表达水平(图4)。高CO2浓度下舞毒蛾雌虫触角中LdOR2表达量与对照组相比分别下降21%和29%(P < 0.05);舞毒蛾雄虫触角中LdOR2与对照组相比分别下降了43%和7%(P < 0.05)。
2.4 不同CO2浓度处理下舞毒蛾LdOR2沉默体行为学分析
注射dsOR2能显著抑制舞毒蛾LdOR2的表达(图5),在397 μL/LCO2浓度下,注射dsOR2的处理组雌雄成虫LdOR2基因的相对表达量比对照组分别显著下降了58.00%和67.00%(P < 0.01)。在550 μL/LCO2浓度处理下,注射dsOR2的雌雄成虫中LdOR2基因的相对表达量与对照组相比,分别显著下降了63.00%和54.00%(P < 0.01)。在750 μL/LCO2浓度处理下,注射dsOR2的雌雄成虫中LdOR2基因的相对表达量比对照组显著下降了40.00%和52.00%(P < 0.01)。
注射dsOR2的舞毒蛾雌虫与对照组相比,对丁香酚、顺-3-己烯-1-醇和α-蒎烯的趋向性明显减弱;注射dsOR2的舞毒蛾雄虫与对照组相比,对丁香酚、石竹烯和顺-3-己烯-1-醇的趋向性减弱,注射dsOR2的舞毒蛾对其他挥发物与对照组无显著性差异。与对照CO2相比,550和750 μL/LCO2下舞毒蛾雌雄成虫对7种挥发性物质的反应率明显降低(图6和图7)。
3. 结论与讨论
在昆虫嗅觉系统信号识别过程中,气味受体(ORs)发挥着关键的作用[19]。昆虫ORs与G蛋白偶联受体结构相似,但其N末端位于细胞膜内侧,C末端位于细胞膜外。舞毒蛾LdOR2基因编码的氨基酸序列分析结果表明,其含有6个跨膜结构,该结果也与棉铃虫(Helicoverpa armigera)HarmOR9、小菜蛾(Plutella xylostella)PxylOR18和中华蜜蜂(Apis cerana cerana)AcerOR113的结果相似[20-22],该结果也印证了Bengtsson等[23]提出的昆虫气味受体可能有4 ~ 8个跨膜结构的推测。由此推断LdOR2符合昆虫气味受体的结构特征。进化树分析表明,舞毒蛾气味受体与夜蛾科(Noctuidae)昆虫气味受体氨基酸序列同源性高,LdOR2与黏虫QNS36221.1和浅灰色夜蛾ALM26210.1亲缘关系近而聚为一类,其中与黏虫QNS36221.1同源性最高,为72.10%。
气味受体的功能与其表达模式紧密相关[24],不同气味受体的表达模式存在差异[25]。本研究舞毒蛾LdOR2基因在雌、雄成虫触角中的表达量显著高于其他组织,这与甜菜夜蛾(Spodoptera exigua)和棉铃虫[26-27]的表达模式类似。气味受体在感觉神经元中发挥作用,而触角作为重要的感器,其上分布着大量感觉神经元[28]。因此,LdOR2在触角中高表达符合气味受体的表达模式,同时LdOR2在雌、雄触角中的表达无显著差异性,这表明其蛋白产物属于普通气味受体。除在触角中高表达外,LdOR2在其他组织中均有表达,这与多音天蚕蛾(Antheraea polyphemus)和斜纹夜蛾(Spodoptera litura)表达模式类似[29-30]。同时验证了普通气味受体具有较大差异的功能分化。此外,在舞毒蛾整个发育阶段均能检测出LdOR2的表达,推测LdOR2可能具有嗅觉感受以外的其他功能。
在全球气候变化的环境下,昆虫对CO2浓度变化的响应极其敏感。前人研究表明,在高CO2浓度下饲养的棉蚜(Aphis gossypii)对植物挥发物的响应更强,对棉苗的趋向性更强[31]。高CO2浓度下埃及伊蚊(Aedes aegypti)对刺激气味源的反应时间更长,影响其寻找寄主的行为活动[32]。本文研究表明,高CO2浓度下舞毒蛾对7种挥发物的反应率均有所下降,说明CO2浓度变化能够影响舞毒蛾对气味的敏感性,影响舞毒蛾的嗅觉反应。CO2浓度变化对昆虫生长发育等表型变化影响的研究较多,而昆虫对这种变化响应的分子机制研究较少。舞毒蛾触角中LdOR2基因的表达在高CO2浓度下被显著抑制,但其在雌、雄触角中受抑制的程度不同。利用RNAi技术结合电生理反应或行为学反应研究昆虫的嗅觉相关基因,能够准确有效地探究目的基因的功能。Liu等[33]通过注射siRNA抑制ORCO基因的表达,导致白纹伊蚊(Aedes albopictus)对寄主偏好性显著降低。Zhou等[34]通过注射siRNA干扰绿盲蝽(Apolygus lucorum)AlucORCO基因,绿盲蝽对丁酸-反-2-己烯酯的反应显著降低。沉默LdOR2的舞毒蛾雌、雄成虫对丁香酚和顺-3-己烯-1-醇的趋向性减弱,说明LdOR2基因可能是识别丁香酚和顺-3-己烯-1-醇气味分子的重要基因,而沉默LdOR2基因后,舞毒蛾雌虫对α-蒎烯的趋向性明显减弱,但在雄成虫中未表现出明显规律,LdOR2基因在识别α-蒎烯是否具有性别差异还有待进一步研究。不同CO2浓度下LdOR2基因沉默对气味的趋性变化不同,可能是由于高CO2浓度下LdOR2基因的表达量受到抑制,LdOR2基因表达水平下降可能影响嗅觉系统的传递效率,进而影响舞毒蛾对气味分子的敏感性。本文为深入研究舞毒蛾的嗅觉机制及舞毒蛾在全球气候变化下的适应性提供了理论基础。
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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。E indicates 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 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)。A, silencing efficiency at 397 μL/LCO2 concentration; B, silencing efficiency at 550 μL/LCO2 concentration; C, silencing efficiency at 750 μL/LCO2 concentration; 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 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。 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
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 Lymantria 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 mass 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) 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 usagedsOR2 TAATACGACTCACTATAGGGAG
GCGATGATCGAAACTTGCTAATACGACTCACTATAGGGCG
GACACAATCATAGTCACCA534 dsRNA合成
dsRNA synthesisdsRed TAATACGACTCACTATAGGG
GAGAACGTCATCACCGAGTTTAATACGACTCACTATAGGG
GATGGTGTAGTCCTCGTTGT658 LdOR2 GAGTTTCGCCGTCAGTCACA CACGCGCATGAACCGTAAAC 201 实时荧光定量
Real time fluorescent quantitationActin 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。Notes: dsOR2 is double-stranded RNA of the LdOR2; dsRed is double-stranded RNA of the control gene. 表 3 试验中所使用的气味源
Table 3 List of odors used in the experiments
化合物名称
Compound nameCAS登录号
CAS registry No.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 -
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