【作者】 李娜；

【导师】 李国清；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2014， 硕士

【摘要】 昆虫有着极发达的嗅觉系统,藉此识别环境中大量的化学信息并做出相应的生理和行为反应,如逃避天敌,寻找食物源、配偶和产卵场所等。在昆虫嗅觉感受器腔内发现两种可溶性蛋白：气味结合蛋白(odorant-binding proteins, OBPs)和化学感受蛋白(chemosensory proteins, CSPs)。CSPs是一类新的蛋白,可能与昆虫识别外界化学信息有关,但其确切的生理功能仍未知。本文基于棉蚜Aphis gossypii转录组数据,克隆得到了7条编码化学感受蛋白(AgosCSP)的cDNA序列,测定了棉蚜化学感受蛋白的时空表达谱,并利用蛋白体外表达技术及荧光竞争结合实验探讨了AgosCSPs的生理功能。结果如下。1.棉蚜化学感受蛋白的基因克隆及序列分析结合转录组数据分析并利用同源性较高的cDNA序列和RACE技术,克隆了棉蚜7个CSP基因的全长cDNA序列。7个基因分别命名为分别命名为AgosCSP1(GenBank登录号：KC017750)、AgosCSP2(GenBank登录号：KC017751)、AgosCSP3(GenBank登录号：KJ451424)、AgosCSP4(GenBank登录号：KJ451425)、AgosCSP8(GenBuck登录号：KC017753)、AgosCSP13(GenBack登录号：FJ387488)、AgosCSP 17(GenBank登录号：KC017754)。序列分析表明,所有CSP均具有典型CSP的序列特征,其中包括4个保守的半胱氨酸位点。2.棉蚜化学感受蛋白动态时空表达分析采用qRT-PCR技术对AgosCSPs在不同时期、不同虫态的棉蚜中mRNA的表达量进行了分析,同时选取秋蚜无翅成虫作为材料研究了AgosCSPs在秋蚜不同组织中的分布情况。试验结果表明AgosCSPs表达分布较广泛,在不同时期不同组织中都有表达,且表达是有差异的。推测其可能执行者各种不同的生理功能。其中AgosCSP1在秋蚜无翅成虫的表达量较高,结合其在足中表达量显著高于其他组织,推测其在寄主定位中有作用；AgosCSP2在越冬寄主木槿树上的棉蚜中相对表达量较其他时期的棉蚜低,在取食棉花的伏蚜和秋蚜中表达量较高,推测该基因与棉蚜对寄主适应性有关。而AgosCSP2在相同时期、寄主相同的棉蚜若虫中相对表达量高于成虫表达量,暗示该基因主要参与棉蚜若虫对寄主的适应过程；AgosCSP3在苗蚜无翅型表达量要显著高于在其他时期,暗示该基因可能涉及对苗蚜无翅成虫和无翅若虫的行为影响。3.棉蚜化学感受蛋白的体外表达及纯化将PCR扩增的AgosCSPs目的产物连接到带有与特异性引物相同酶切位点的表达载体pGEX-4T-1中,构建pGEX-4T-1/AgosCSPs重组表达载体。并选择BL21(DE3)作为表达菌株,通过IPTG诱导进行原核表达。经分析,表达出的蛋白全部为可溶性,经亲和层析、凝血酶处理后纯化出6个高纯度的棉蚜CSP蛋白。4.棉蚜化学感受蛋白与配体结合能力分析利用荧光竞争结合实验的方法测定了棉蚜CSP蛋白对包括棉蚜报警信息素在内的85种化学信息素的结合能力。试验结果表明仅有AgosCSP1与1-氨基蒽和β-紫罗兰酮较强的结合能力,结合常数Ki值分别为2.43 μmol·L-1和22.67 μmol·L-1推测其在寄主定位中起一定的作用。而其他蛋白对不同的气味物质有一定的结合能力,但结合能力都很弱,推测部分AgosCSPs承担着除嗅觉作用以外的更为复杂的生理功能,从而调节调节昆虫的生理节奏或者生长发育。更多还原

【Abstract】 Insects possess sensitive chemosensory systems that recognize a variety of chemical compounds. Using these sensitive olfactory systems, insects detect their environment to identify enemies, to find food sources, to recognize mates and to locate oviposition sites. In insects, two main classes of polypeptides have been identified in the lymph of chemosensilla:odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). CSPs may represent a new class of soluble carrier proteins involved in insect chemoreception. However, the physiological function of CSPs remains unclear. In this study, seven full-length cDNAs encoding chemosensory proteins were cloned from Aphis gossypii. In order to understand the role of CSPs in Aphis gossypii, quantitative real-time polymerase chain reaction (qPCR) was used to determine the expression patterns of the 7 CSPs genes. Finally, a varity of volatile chemicals were used to analyze the binding properties in competitive binding assays.1. Molecular cloning and sequence analysis of AgosCSP genesBased on the transcriptomic data and using degenerate PCR, seven full-length CSP cDNAs were identified from Aphis gossypii, which were respectively named as AgosCSPl (GenBank accession no. KC017750), AgosCSP2 (GenBank accession no. KC017751), AgosCSP3 (GenBank accession no. KJ451424), AgosCSP A (GenBank accession no. KJ451425), AgosCSP8 (GenBank accession no. KC017753), AgosCSP13 (GenBank accession no. FJ387488) and AgosCSP17(GenBank accession no.KC017754). Sequence analysis showed that all the 7 CSPs share typical characteristics in CSPs. Each member had four conserved cysteines.2. Expression profiles of AgosCSPsThe expression levels of AgosCSPs in different ecologically developmental stages, different forms, and various tissues in the apterous autumn adults were examined by RT-PCR. The results indicated that the CSPs were widely expressed in different stages and various tissues. Such expression profiles indicated important chemosensory and non-chemosensory functions. AgosCSPl had a higher expression level in the alatae autumn adults and in the legs, indicating its role in host recognition. The expression levels of AgosCSP1 in nymphs were significantly higher than those in adult. Moreover, it was highly expressed in the aphids from cotton than in the aphids from overwintering host hibiscus. These results suggest that the AgosCSP1 gene is most likely involed in the adaptation of nymphs to their hosts. Furthermore, AgosCSP3 was highly expressed in the alatae aphids from cotton, indicating its role in the behavior of alatae aphids on the cotton hosts.3. Prokaryotic expression and purification of AgosCSPsIn order to study the function of CSPs in the chemical communication system, the AgosCSP proteins were expressed using a prokaryotic expression system. The AgosCSP genes were individually introduced into the pGEX-4T-1 expression vector, and then were expressed in the BL21(DE) Escherichia coli after the induction of IPTG. SDS-PAGE analysis revealed that all the expressed AgosCSPs were soluble. These recombinant proteins were purified through affinity chromatography, and the GST-tags were removed by enzyme digestion. Finally, six AgosCSP proteins were obtained.4. Ligand-binding affinity analyses of AgosCSPThe binding affinities of the 6 AgosCSPs were tested by fluorescence competitive binding experiment with 85 odorant compounds including the alarm pheromones. AgosCSPl showed high binding affinities to 1-aminoanthracene (2.43 μmol·L-1) and P-ionone (22.67 pmol·L-1). Since 1-aminoanthracene is one of plant volatiles and　β-ionone is a volatile widerly distributed in flowers and other tissues of many plant species, it is suggested that AgosCSP1 is involved in the host recognition. However, the remaining 5 AgosCSPs had low binding affinities to all the 85 odorant compounds. They may play other roles rather than olfaction in A. gossypii.更多还原