Santamaría María E, González-Cabrera Joel, Martínez Manuel, Grbic Vojislava, Castañera Pedro, Díaz Lsabel, Ortego Félix
Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, 28040 Madrid, Spain; Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus Montegancedo, Autovía M40 (Km 38), 28223 Pozuelo de Alarcón, Madrid, Spain; Department of Biology WSC 339/341, The University of Western Ontario, 1151 Richmond St, London, ON N6A 5B7, Canada.
Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, 28040 Madrid, Spain.
J Insect Physiol. 2015 Jul;78:69-77. doi: 10.1016/j.jinsphys.2015.05.002. Epub 2015 May 7.
Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC-nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.
通过比较植食性螨类二斑叶螨（Tetranychus urticae）体内和粪便提取物中的活性，对其消化蛋白酶进行了表征。在螨体和粪便中均检测到了天冬氨酸蛋白酶、组织蛋白酶B样和L样以及豆荚天冬氨酸蛋白酶活性，其中天冬氨酸蛋白酶和组织蛋白酶L样蛋白酶在螨粪便中的比活性分别比在螨体中高约5倍和2倍。一般来说，所有这些活性的维持都与饲养螨类的宿主植物（豆类、番茄或玉米）无关。值得注意的是，这是首次在植食性螨类中报道豆荚天冬氨酸蛋白酶样活性，其特征在于能够水解特定底物Z-VAN-AMC，被二硫苏糖醇（DTT）激活并被吲哚-3-乙酸（IAA）抑制，但不被E-64抑制。对螨粪便进行的无凝胶纳米液相色谱-纳米电喷雾-四极杆飞行时间串联质谱（nanoLC-nanoESI-QTOF MS/MS）蛋白质组分析，鉴定出了四种组织蛋白酶L和一种天冬氨酸蛋白酶（在该物种基因组中总共鉴定出29种组织蛋白酶L、27种组织蛋白酶B、19种豆荚天冬氨酸蛋白酶和两种天冬氨酸蛋白酶基因）。基因表达分析表明，在螨粪便中鉴定出的四种组织蛋白酶L和天冬氨酸蛋白酶，以及在粪便中未检测到的两种组织蛋白酶B和两种豆荚天冬氨酸蛋白酶，相对于胚胎而言，在叶螨的取食阶段（幼虫、若虫和成虫）表达水平较高。综上所述，这些结果表明半胱氨酸蛋白酶和天冬氨酸蛋白酶在二斑叶螨中具有消化作用。在我们的研究中，分析的组织蛋白酶B和L、豆荚天冬氨酸蛋白酶和天冬氨酸蛋白酶基因的表达，在雌性成虫取食过表达HvCPI-6胱抑素（特异性靶向组织蛋白酶B和L）或靶向丝氨酸蛋白酶的CMe胰蛋白酶抑制剂的拟南芥植物后有所增加。这种非特异性反应表明，除了补偿被抑制剂靶向的酶之外，二斑叶螨消化蛋白酶表达的增加可能作为抵御摄入的植物防御蛋白的第一道屏障。
