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1.
【目的】触角结合蛋白(antennal binding proteins,ABPs)为昆虫气味结合蛋白(odorant binding proteins,OBPs)家族的一个亚类,是昆虫识别和响应外界环境中气味信号的载体之一,对昆虫的生存和繁衍有着重要的意义。明确触角结合蛋白在小菜蛾Plutella xylostella(L.)嗅觉识别中的作用,有助于揭示小菜蛾嗅觉识别分子机制。【方法】利用PCR技术克隆小菜蛾的一个触角结合蛋白基因;采用实时荧光定量PCR技术对该基因在小菜蛾不同发育阶段和成虫不同组织中的表达量进行分析;利用荧光竞争结合实验测试该触角结合蛋白与39种配基化合物的结合特性。【结果】成功克隆了一个小菜蛾触角结合蛋白基因,命名为Pxyl OBP31(Gen Bank登录号:KT156676)。序列分析结果显示,其开放阅读框全长411 bp,编码136个氨基酸,N端自起始位置开始21个氨基酸为信号肽,含有气味结合蛋白家族的6个保守半胱氨酸残基,预测分子量为14.74 k D,等电点为4.41。表达谱分析表明,Pxyl OBP31主要在雄蛾中表达,且交配后的雄蛾中表达量明显降低;该基因在小菜蛾触角中有较高表达,在雄蛾触角中的表达量比雌蛾触角中高近2倍。结合特性实验结果显示,Pxyl OBP31与醛、酮、萜品油烯以及邻苯二甲酸二异丁酯等物质的结合能力较强,与3种性信息素及其他烯烃与酯类结合能力弱。【结论】本研究明确了Pxyl OBP31的核苷酸序列以及发育和组织表达谱。根据qRT-PCR和荧光竞争结合实验结果,推测Pxyl OBP31蛋白可能与小菜蛾觅偶、定位寄主植物等行为有关。  相似文献   

2.
利用RT-PCR技术克隆了光肩星天牛Anoplophora glabripennis Motschulsky气味结合蛋白(odorant binding proteins,OBPs)基因AglaOBP1(Gen Bank登录号:KX660670),AglaOBP1的开放阅读框长435 bp,编码144个氨基酸,其中N端有21个氨基酸组成的信号肽序列,成熟蛋白序列具有4个保守的半胱氨酸残基,AglaOPB1属于Minus-C OBP亚家族基因。AglaOBP1主要在成虫触角中表达,且雌虫触角中的表达量显著高于雄虫触角。重组蛋白AglaOBP1与34种气味配体的竞争结合实验表明,AglaOBP1具有广泛的结合谱,能与五角枫挥发物中的醇类、醛类、萜烯类和酮类物质结合,其中与顺-3-己烯-1-醇、顺-2-己烯-1-醇、1-己醇、反-2-己烯醛、反-2-癸烯醛、β-石竹烯、(+)-桧烯、α-蒎烯、1-(2,3-二甲基苯基)-乙酮的结合能力较强,结合常数分别为5.88、8.33、12.29、12.55、11.90、7.47、9.07、10.29和13.12μM。此外,配体的官能团、碳链长度、空间构型也影响AglaOBP1对气味分子的结合。AglaOBP1基因在成虫触角中高丰度表达,其重组蛋白能够与五角枫挥发物的多种组分结合,表明AglaOBP1在成虫寄主定位选择中起重要作用。  相似文献   

3.
绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性   总被引:4,自引:0,他引:4  
气味结合蛋白(odorant binding proteins, OBPs) 在昆虫嗅觉识别中起着重要的作用, 尤其是在运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体(olfactory receptors, ORs)的过程中发挥关键作用。明确OBPs在昆虫同外界进行信息交流过程中的作用有利于阐明昆虫嗅觉识别的机制, 同时可为利用干扰昆虫嗅觉识别来进行害虫防治奠定理论基础。本研究克隆了一个绿盲蝽Apolygus lucorum (Meyer-Dür)气味结合蛋白AlucOBP7基因(GenBank登录号: JQ675724), 并进行了原核表达, 以1-NPN为荧光探针采用荧光竞争结合实验研究了AlucOBP7蛋白和10种棉花挥发物及 6种性信息素类似物的结合能力。结果表明: 在10种棉花挥发物中, AlucOBP7能够和2 己酮及水杨酸甲酯有效结合, 结合常数分别为55.13 μmol/L和28.26 μmol/L。在6种盲蝽性信息素类似物中, 4-氧代-反-2-己烯醛和AlucOBP7 具有较强的结合能力, 结合常数为23.14 μmol/L。丁酸乙酯、 丁酸丁酯及己酸己酯也能够和AlucOBP7 有效结合, 但结合能力中等, 结合常数分别为30.58, 39.26和35.81 μmol/L。初步推测, AlucOBP7 可能是绿盲蝽性信息素结合蛋白(pheromone binding proteins, PBPs), 并在感受性信息素和植物挥发物的过程中发挥双重功能。  相似文献   

4.
【目的】为了更好地了解昆虫气味结合蛋白(odorant binding proteins, OBPs)在梨小食心虫Grapholita molesta(Busck)嗅觉识别中的作用,并明确其与寄主挥发物的结合特性。【方法】利用RT-PCR和RACE技术克隆梨小食心虫OBP基因;采用RT-PCR和实时定量PCR对该基因在成虫不同组织和羽化后不同日龄成虫中的表达情况进行了测定;以N-phenyl-1-naphthylamine(1-NPN)为荧光探针,采用荧光竞争结合试验对GmolOBP3蛋白的结合特性进行了分析。【结果】得到梨小食心虫一个新的气味结合蛋白基因,命名为GmolOBP3(GenBank登录号:KF395363)。GmolOBP3开放阅读框全长492 bp,编码163个氨基酸残基,预测分子量和等电点分别为18.72 kDa和4.93,呈酸性,具有典型的6个半胱氨酸位点。GmolOBP3在雌、雄成虫触角和腹部均有表达,成虫在羽化后5 d内,雌蛾触角中GmolOBP3表达量随羽化后日龄而增加,但雄蛾在羽化后第5天触角中 GmolOBP3表达量显著降低。通过构建GmolOBP3原核表达载体,在大肠杆菌Escherichia coli中诱导表达并获得了GmolOBP3重组蛋白。荧光竞争结合实验对GmolOBP3蛋白与16种寄主挥发物及4种性信息素类似物的结合力发现,在供试的4种梨小食心虫性信息素类似物中,GmolOBP3蛋白与反-8-十二碳烯醋酸酯和十二烷-1-醇不结合,而与顺-8-十二碳烯醋酸酯和顺-8-十二碳烯醇结合,但结合力较弱,结合常数分别为83.00和103.70 μmol/L;与16种寄主挥发物结合能力也不强,其中结合最强的是β 紫罗酮,结合常数为49.36 μmol/L。【结论】由此推断,GmolOBP3具有选择性识别和结合各种配基的特性。  相似文献   

5.
二化螟Minus-C气味结合蛋白的分子克隆及功能鉴定   总被引:3,自引:0,他引:3  
气味结合蛋白(odorant binding proteins, OBPs)在昆虫对寄主气味的感受中起重要作用, 但有关Minus-C OBP及其功能的报道很少。本研究通过基因组数据分析并利用RACE技术, 克隆和鉴定了二化螟Chilo suppressalis (Walker)的一个Minus-C OBP基因, 命名为CsupOBP1(GenBank登录号: KC492498)。CsupOBP1基因的开放阅读框长423 bp, 编码141个氨基酸, 其中N端的18个氨基酸为预测的信号肽序列, 成熟蛋白序列中具有4个保守的半胱氨酸位点。实时定量PCR分析显示, 该基因在幼虫头部及成虫雌雄足、 翅和雄性触角等化感组织中高表达, 其中在雄虫触角内的表达量显著高于雌虫触角。利用荧光竞争结合实验对CsupOBP1重组蛋白与38种化合物的结合特性的测定表明, 重组CsupOBP1与β 紫罗兰酮的结合能力最强(Ki=9.53 μmol/L)。触角电位测定表明, 二化螟成虫可对β-紫罗兰酮产生显著的触角电生理反应, 但雄虫反应明显强于雌虫, 与结合试验及雄虫触角中CsupOBP1的表达量显著高于雌虫触角的测定结果相一致。鉴于β-紫罗兰酮是水稻等植物中普遍存在的一种芳香气味组分, 推测CsupOBP1可能通过对该气味的结合和运输, 从而在二化螟对寄主植物的嗅觉定向中起作用。  相似文献   

6.
【目的】克隆和鉴定光肩星天牛Anoplophora glabripennis气味结合蛋白(odorant binding proteins,OBPs)基因,明确其表达特点及与寄主植物挥发物的结合特性,有助于阐明光肩星天牛嗅觉识别的分子机制。【方法】根据光肩星天牛雌成虫触角转录组数据,利用RT-PCR克隆OBP12基因,并进行生物信息学分析。通过实时定量PCR(qRT-PCR)测定OBP12在光肩星天牛成虫触角、头(移除触角)、胸、腹、足、翅中的转录水平。利用原核表达系统和Ni离子亲和层析技术表达和纯化OBP12重组蛋白,荧光竞争结合实验测定重组蛋白与39种气味配体的结合能力。【结果】获得光肩星天牛气味结合蛋白基因AglaOBP12(GenBank登录号:KX890109)的完整编码序列,其开放阅读框长414 bp,编码137个氨基酸,N末端具有18个氨基酸组成的信号肽序列,蛋白序列具有6个保守的半胱氨酸残基,Agla OPB12属于Classical OBPs亚家族基因。qRT-PCR测定结果表明,AglaOBP12主要在成虫触角中表达,在其他组织微量表达。在待测的39种寄主植物挥发物中,重组蛋白AglaOBP12仅与19种化合物具有结合活性,表明AglaOBP12对寄主植物挥发物具有明显的选择结合特性。重组蛋白AglaOBP12与十二烷醇、十四烷醇、法尼醇、十二醛、乙酸-顺-3-己烯酯和β-石竹烯的结合能力较强,结合常数分别为1.96,0.96,1.03,0.82,0.77和0.74μmol/L。【结论】明确了AglaOBP12的核苷酸和氨基酸序列组成,重组AglaOBP12蛋白与主链有12个碳原子的醇类、醛类和萜烯类挥发物有特异性的结合活性。根据AglaOBP12基因的表达特点和重组蛋白的结合特性,推测AglaOBP12在光肩星天牛成虫定位补充营养寄主植物中发挥重要作用。  相似文献   

7.
【目的】为了更好地了解性信息素结合蛋白(pheromone binding proteins, PBPs)在桃蛀螟Conogethes punctiferalis (Guenée)嗅觉识别过程中的作用,明确其与配体化合物的结合特性。【方法】本研究利用RT-PCR结合RACE方法克隆了桃蛀螟一个性信息素结合蛋白基因;采用Real-time PCR方法分析了该蛋白在桃蛀螟不同发育阶段及雌雄蛾间的表达差异;利用荧光竞争结合实验对Cpun-PBP1蛋白与16种配基化合物的结合特性进行了分析。【结果】克隆了一个桃蛀螟性信息素结合蛋白基因,命名为Cpun-PBP 1(GenBank登录号:KP027486)。Cpun-PBP 1开放阅读框全长510 bp,编码 169个氨基酸,预测分子量为19.12 kDa,等电点为5.09,N-末端包括由起始位置开始的30个氨基酸组成的信号肽。蛋白特征分析显示,该氨基酸序列具有昆虫气味结合蛋白的典型特征,即含有6个保守的半胱氨酸残基。Cpun-PBP 1在桃蛀螟成虫阶段表达量最高,且几乎全部在触角中表达,卵期微量表达,幼虫期和蛹期均不表达。通过构建Cpun-PBP 1原核表达载体,诱导并获得Cpun-PBP 1重组蛋白。荧光竞争结合实验对2种性信息素组分和14种寄主植物挥发物的结合力发现,Cpun-PBP1不但能有效地与桃蛀螟性信息素组分(顺-10-十六碳烯醛和十六醛)结合,结合常数分别为7.32和9.39 μmol/L;还能与8种寄主植物挥发物有效结合;其中,与莰烯的结合能力最强,结合常数为3.76 μmol/L。【结论】根据这些结果,我们推测Cpun-PBP1在桃蛀螟感受性信息素和寄主植物挥发物的过程中发挥着双重作用。  相似文献   

8.
茶谷蛾(Agriophara rhombata Meyr.)触角上表达的嗅觉相关基因是其交配、寻找食物来源、寻找产卵地、避免有害化合物等行为的重要分子基础。为了研究茶谷蛾触角的嗅觉机制,分析了雌雄蛾触角功能差异,并筛选触角气味结合蛋白基因,本研究采用转录组测序和实时定量PCR技术,对茶谷蛾雌、雄蛾触角的基因表达情况进行了分析,共注释到37 666个基因,其中雌、雄蛾共有的表达基因数目为34 720个,雌蛾特有表达基因有1 739个,雄蛾特有表达基因有1 207个。在雌、雄蛾触角中共鉴定到170个化学感受相关基因,包括33个气味结合蛋白基因(odorant binding protein genes,OBPs), 42个味觉受体基因(gustatory receptor genes,GRs), 12个化学感受蛋白基因(chemosensory protein genes,CSPs), 52个嗅觉受体基因(olfactory receptor genes,ORs), 27个离子型受体基因(ionotropic receptor genes,IRs), 4个感觉神经元膜蛋白基因(sensor...  相似文献   

9.
苜蓿盲蝽气味结合蛋白基因Alin-OBP1的克隆及表达谱分析   总被引:2,自引:0,他引:2  
有证据表明昆虫气味结合蛋白(odorant binding proteins, OBPs)与其嗅觉识别密切相关,起着运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体的关键作用.为了更好地了解OBPs在苜蓿盲蝽Adelphocoris lineolatus (Goeze)嗅觉识别中的作用,本研究首次克隆了苜蓿盲蝽气味结合蛋白基因Alin-OBP1 (GenBank序列号GQ477022). 测序和序列分析结果表明,该基因开放阅读框长438 bp, 编码145个氨基酸,预测分子量为15.69 kDa,等电点为5.01,N-末端疏水区包含由18个氨基酸组成的信号肽.蛋白特征分析表明,该基因翻译后的蛋白质具有昆虫气味结合蛋白的典型特征,即氨基酸序列中有6个保守的半胱氨酸残基.利用RT-PCR和Real-time PCR技术对Alin OBP1在苜蓿盲蝽成虫不同组织和各个发育阶段的表达水平进行了测定,结果显示Alin-OBP1几乎全部在触角中表达.不同发育阶段Alin-OBP1表达量不同,在5龄若虫和成虫阶段表达水平最高.结果提示Alin-OBP1可能在苜蓿盲蝽感受包括性信息素在内的外界化合物的过程中发挥着重要作用.  相似文献   

10.
【目的】本研究旨在明确气味结合蛋白(odorant binding proteins, OBPs)在桃蛀螟Conogethespunctiferalis化学感受过程中的生理功能,为以OBPs蛋白为防治靶标的桃蛀螟绿色防控提供理论依据。【方法】基于前期桃蛀螟触角转录组测序数据,利用PCR技术从桃蛀螟触角中获得桃蛀螟气味结合蛋白基因CpunOBP3和CpunOBP4的cDNA序列,采用生物信息学软件分析其核苷酸和氨基酸序列;构建重组表达载体pET-30a/CpunOBP3和pET-30a/CpunOBP4,原核表达并纯化获得重组目的蛋白CpunOBP3和CpunOBP4;最后利用荧光竞争结合实验测定了重组蛋白CpunOBP3和CpunOBP4对24种配体的结合能力。【结果】克隆获得桃蛀螟气味结合蛋白基因CpunOBP3(GenBank登录号: GEDO010000010.1),开放阅读框全长387 bp,编码128个氨基酸,预测分子量大小为14.72 kD;CpunOBP4(GenBank登录号: GEDO010000011.1)开放阅读框全长438 bp,编码145个氨基酸,去除信号肽的CpunOBP4蛋白预测分子量大小为12.82 kD。CpunOBP3和CpunOBP4均具有OBPs的典型特征,即含有6个保守的半胱氨酸残基。CpunOBP3和CpunOBP4重组蛋白均以包涵体形式存在。荧光竞争结合实验发现,CpunOBP3重组蛋白能与测试的7种植物挥发物有效结合,尤其与3-蒈烯的结合能力最强,解离常数Ki值为10.33 μmol/L,但不能与测试的2种桃蛀螟性信息素有效结合;而CpunOBP4重组蛋白既可以与测试的2种性信息素(顺-10-十六碳烯醛和十六醛)结合(解离常数Ki值分别为14.65 μmol/L和7.83 μmol/L),又可以与测试的8种植物挥发物有效结合,尤其与丁酸乙酯的结合能力最强,解离常数Ki值为4.32 μmol/L。【结论】根据这些结果,我们推测CpunOBP3主要在桃蛀螟寄主定位与寄主转移过程中发挥重要作用,而CpunOBP4在桃蛀螟识别性信息素和寄主植物挥发物过程中发挥双重作用。研究结果为利用干扰桃蛀螟嗅觉感受从而调控其发生与危害提供了理论依据。  相似文献   

11.
12.
Chemoreception is a key feature in selection of host plants by insects. We performed a preliminary functional characterization of olfactory proteins isolated from an antennal cDNA library of Monochamus alternatus. We identified four olfactory genes, including two encoding putative classic odorant‐binding proteins (OBPs) and two encoding minus‐C OBPs. We expressed two of the four OBPs, MaltOBP3 and MaltOBP5, in a bacterial system and assessed their ligand specificity by measuring the competitive binding of fluorescent probe, N‐phenyl‐1‐naph‐thylamine, in the presence of 17 volatile beetle‐ or host‐plant‐related ligands. The results indicated that although MaltOBP3 and MaltOBP5 bound a distinctly different group of competitors, both had relatively high binding affinities (Ki < 20 μm ) for certain compounds. The differences in their binding affinities towards host‐plant ligands suggest the roles of MaltOBP3 and MaltOBP5 in host‐plant selection.  相似文献   

13.
Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.  相似文献   

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The soybean aphid, Aphis glycines, is an extreme specialist and an important invasive pest that relies on olfaction for behaviors such as feeding, mating, and foraging. Odorant‐binding proteins (OBPs) play a vital role in olfaction by binding to volatile compounds and by regulating insect sensing of the environment. In this work we used rapid amplification of complementary DNA ends technology to identify and characterize 10 genes encoding A. glycines OBPs (AglyOBPs) belonging to 3 subfamilies, including 4 classic OBPs, 5 Plus‐C OBPs, and one Minus‐C OBP. Quantitative real‐time polymerase chain reaction demonstrated variable specific expression patterns for the 10 genes based on developmental stage and aphid tissue sampled. Expression levels of 7 AglyOBPs (2, 3, 4, 5, 7, 9, and 10) were highest in the 4th instar, indicating that the 4th nymphal instar is an important developmental period during which soybean aphids regulate feeding and search for host plants. Tissue‐specific expression results demonstrated that AglyOBP2, 7, and 9 exhibited significantly higher expression levels in antennae. Meanwhile, ligand‐binding analysis of 5 OBPs demonstrated binding of AglyOBP2 and AglyOBP3 to a broad spectrum of volatiles released by green leaf plants, with bias toward 6‐ to 8‐carbon chain volatiles and strong binding of AglyOBP7 to transβ‐farnesene. Taken together, our findings build a foundation of knowledge for use in the study of molecular olfaction mechanisms and provide insights to guide future soybean aphid research.  相似文献   

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Sitodiplosis mosellana, a periodic but devastating wheat pest, relies on wheat spike volatiles as a cue in selecting hosts for oviposition. Insect odorant‐binding proteins (OBPs) are thought to play essential roles in filtering, binding and transporting hydrophobic odorant molecules to specific receptors. To date, the molecular mechanisms underlying S. mosellana olfaction are poorly understood. Here, three S. mosellana antenna‐specific OBP genes, SmosOBP11, 16 and 21, were cloned and bacterially expressed. Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were investigated using fluorescence competitive binding assays. Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily. Ligand‐binding analysis showed that all three SmosOBPs preferentially bound alcohol, ester and ketone compounds, and SmosOBP11 and 16 also selectively bound terpenoid compounds. In particular, the three SmosOBPs had high binding affinities (Ki < 20 μmol/L) to 3‐hexanol and cis‐3‐hexenylacetate that elicited strong electroantennogram (EAG) response from female antennae. In addition, SmosOBP11 displayed significantly higher binding (Ki < 8 μmol/L) than SmosOBP16 and 21 to 1‐octen‐3‐ol, D‐panthenol, α‐pinene and heptyl acetate which elicited significant EAG response, suggesting that SmosOBP11 plays a major role in recognition and transportation of these volatiles. These findings have provided important insight into the molecular mechanism by which S. mosellana specifically recognizes plant volatiles for host selection, and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping.  相似文献   

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昆虫嗅觉相关蛋白的结构和功能   总被引:2,自引:0,他引:2  
昆虫在长期进化的过程中形成了复杂的嗅觉系统,气味剂结合蛋白(odorant binding proteins,OBPs)、嗅觉受体(olfactory receptors,ORs)是其最主要的组分.其主要作用是结合外围挥发性的气味分子并将信号传递给细胞内的第二信使.OBPs和ORs的结构、功能、表达、进化是昆虫行为与进化关系的重要研究领域和研究热点.本文主要总结了近年来昆虫OBPs和ORs的结构特点、生理功能、表达特点、遗传进化等方面研究的最新进展,对OBPs和ORs的研究趋势进行了展望,为昆虫嗅觉系统进化研究及寻找害虫防治新途径提供参考信息.  相似文献   

20.

Background

The complex societies of ants and other social insects rely on sophisticated chemical communication. Two families of small soluble proteins, the odorant binding and chemosensory proteins (OBPs and CSPs), are believed to be important in insect chemosensation. To better understand the role of these proteins in ant olfaction, we examined their evolution and expression across the ants using phylogenetics and sex- and tissue-specific RNA-seq.

Results

We find that subsets of both OBPs and CSPs are expressed in the antennae, contradicting the previous hypothesis that CSPs have replaced OBPs in ant olfaction. Both protein families have several highly conserved clades with a single ortholog in all eusocial hymenopterans, as well as clades with more dynamic evolution and many taxon-specific radiations. The dynamically evolving OBPs and CSPs have been hypothesized to function in chemical communication. Intriguingly, we find that seven members of the conserved clades are expressed specifically in the antennae of the clonal raider ant Cerapachys biroi, whereas only one dynamically evolving CSP is antenna specific. The orthologs of the conserved, antenna-specific C. biroi genes are also expressed in antennae of the ants Camponotus floridanus and Harpegnathos saltator, indicating that antenna-specific expression of these OBPs and CSPs is conserved across ants. Most members of the dynamically evolving clades in both protein families are expressed primarily in non-chemosensory tissues and thus likely do not fulfill chemosensory functions.

Conclusions

Our results identify candidate OBPs and CSPs that are likely involved in conserved aspects of ant olfaction, and suggest that OBPs and CSPs may not rapidly evolve to recognize species-specific signals.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-718) contains supplementary material, which is available to authorized users.  相似文献   

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