豌豆蚜唾液蛋白家族的克隆及在不同发育阶段的表达

豌豆蚜Acyrthosiphon pisum是一种重要的刺吸式害虫, 其分泌的唾液对取食寄主植物和传播植物病毒有重要的作用。为了探讨蚜虫唾液蛋白的功能, 本研究克隆了在豌豆蚜唾液腺高表达的一个未知功能的蛋白家族, 该家族包括13个基因, 编码14种蛋白, 其中4个基因在唾液腺高表达。这个家族是蚜虫特有的蛋白家族, 富含半胱氨酸, 有14个半胱氨酸高度保守, 其中6个半胱氨酸形成3个保守的CXXC结构域。通过与基因组比对, 发现这个家族的基因没有内含子, 分布在基因组的9个scaffold上。用半定量逆转录PCR检测了每个成员在豌豆蚜不同发育阶段的表达, 结果显示这个家族没有发育阶段特异性。推测这个家族的表达可能具有组织特异性, 有氧化还原酶或DNA甲基化酶的功能。     

Characterization of a novel salivary immunosuppressive protein from Ixodes ricinus ticks.

In tick salivary glands, several genes are induced during the feeding process, leading to the expression of new proteins. These proteins are typically secreted in tick saliva and are potentially involved in the modulation of the host immune and hemostatic responses. In a previous study, the construction and the analysis of a subtractive library led to the identification of Ixodes ricinus immunosuppressor (Iris), a novel protein, differentially expressed in I. ricinus salivary glands during the blood meal. In the present study, the data strongly suggest that this protein is secreted by tick salivary glands into the saliva. In addition, Iris is also found to modulate T lymphocyte and macrophage responsiveness by inducing a Th2 type response and by inhibiting the production of pro-inflammatory cytokines. In conclusion, these results suggest that Iris is an immunosuppressor, which might play an important role in the modulation of host immune response.     

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

Identification of novel tick salivary gland proteins for vaccine development

Methods currently used to control Ixodes scapularis ticks rely principally on acaricidal applications which suffer from a number of limitations. Recently, host vaccination against ticks has been shown to be a promising alternative tick control method. In tick salivary glands, numerous genes are induced during the feeding process. Many of these newly expressed proteins are secreted in tick saliva and may play a role in modulating host immune responses and pathogen transmission. We have performed suppression subtraction hybridization to identify unique I. scapularis salary gland proteins specifically expressed during engorgement. We have cloned and sequenced ten unique salivary gland-associated cDNAs that are up-regulated during feeding. The protein products of these genes represent potential vaccine candidates for use in the control of ticks and to prevent transmission of tick-borne diseases.     

棉铃虫幼虫唾液腺cDNA文库的构建及EST分析

棉铃虫Helicoverpa armigera (Hübner)幼虫唾液中的各种酶类及各种生化组分在棉铃虫与植物相互作用及协同进化中起到重要作用; 唾液腺是棉铃虫唾液成分的合成器官。本研究通过构建棉铃虫幼虫唾液腺全长cDNA文库, 测序得到1 502条EST序列, 聚类分析后获得821个unigenes, 为筛选棉铃虫与寄主互作信号因子提供基因信息资源。使用Blast2 GO软件对821个unigenes进行了比对和功能注释, 初步获得棉铃虫幼虫唾液腺中mRNA的构成特征。结果显示, 在棉铃虫唾液腺ESTs文库中, 鉴定得到脂类相关消化酶基因17个, 糖类相关消化酶基因5个, 半胱氨酸蛋白酶基因1个, 丝氨酸蛋白酶基因20个（其中16个为新发现）, 提示唾液腺的主要功能是分泌消化酶进行预消化; 还发现在棉铃虫幼虫唾液腺中存在表皮蛋白、 气味结合蛋白和化学感受蛋白基因。结果为研究棉铃虫预消化系统打下基础。     

Mining the secretome of the root-knot nematode Meloidogyne chitwoodi for candidate parasitism genes

Parasite proteins secreted at the interface of nematode and host are believed to play an essential role in parasitism. Here, we present an efficient pipeline of bio-informatic algorithms and laboratory experiments to identify candidate parasitism genes within nematode secretomes, i.e. the repertoire of secreted proteins in an organism. We performed our approach on 12 218 expressed sequence tags (ESTs) originating from three life stages of the plant parasitic nematode Meloidogyne chitwoodi —a molecularly unexplored root-knot nematode species. The ESTs from M. chitwoodi were assembled into 5880 contigs and open reading frames translated from the consensus sequences were searched for features of putative signal peptides for protein secretion and trans-membrane regions, resulting in the identification of 398 secretome members. The products of parasitism genes are secreted by a range of organs, including the oesophageal, amphidial and rectal glands, the intestine, and the hypodermis. To localize the site of expression in M. chitwoodi , we subjected the most abundant secretome members to in situ hybridization microscopy. We found hybridization of one tag in the dorsal oesophageal gland, seven in the two subventral oesophageal glands, two in the intestine and one tag hybridized to the tail tip in the proximity of the phasmids. Four sequences showed similarity to putative parasitism genes from other nematode species, whereas seven represented pioneering sequences. Our approach presents an efficient method to identify candidate parasitism genes, which does not require sophisticated cDNA isolation and selection protocols, and can therefore be used as a powerful starting point for the molecular investigation of parasites.