Shi Min, Chen Xiao-Yu, Zhu Ni, Chen Xue-Xin
Ministry of Agriculture Key Lab of Agricultural Entomology, Institute of Insect Sciences, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China.
Ministry of Agriculture Key Lab of Agricultural Entomology, Institute of Insect Sciences, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China
J Insect Sci. 2014 Jan 1;14:179. doi: 10.1093/jisesa/ieu041. Print 2014.
The phenoloxidase (PO) activation system plays an important role in insect innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating protease (PAP), which is the direct activator of prophenoloxidase (proPO). Despite their importance in the insect PO activation system, content of studies is limited. In this article, we identify two complementary DNAs (cDNAs), PxPAPa and PxPAPb, encoding possible PAPs, from immunized larval hemocytes of the diamondback moth, Plutella xylostella (L.), by RACE method. PxPAPa is 1,149-bp long and encodes a 382-residue open reading frame (ORF) with a predicted 17-residue signal peptide, a clip domain, and a Tryp_Spc domain. PxPAPb is 1,650-bp long and encodes a 440-residue ORF with a predicted 20-residue signal peptide, two clip domains, and a Tryp_Spc domain. PxPAPa and PxPAPb have a high sequence similarity to Manduca sexta (L.) PAP1 and PAP3, respectively. We also examined the transcript patterns of PxPAPa, PxPAPb, and pxPAP3, another clip-domain serine protease gene, response to different microbial challenges by using real-time quantitative polymerase chain reaction. The results show that the transcript abundance of PxPAPa is significantly increased by Micrococcus luteus and Escherichia coli but not Candida albicans. PxPAPb is induced only by Mi. luteus, whereas pxPAP3 could be induced by all the microbes in the test, but the transcript patterns of Mi. luteus, E. coli, and C. albicans are completely different. This study provides new insights into the molecular events that occur during the immune response, particularly melanization cascade that is involved in encapsulation and nodulation of pathogen or parasite invaders via hemocytes in host insects.
酚氧化酶（PO）激活系统在昆虫先天免疫中发挥着重要作用，尤其是在伤口愈合和病原体防御方面。该系统的一个关键成员是前酚氧化酶激活蛋白酶（PAP），它是前酚氧化酶（proPO）的直接激活剂。尽管它们在昆虫PO激活系统中很重要，但相关研究的内容有限。在本文中，我们通过RACE方法从小菜蛾（Plutella xylostella (L.)）免疫幼虫的血细胞中鉴定出两个互补DNA（cDNA），即PxPAPa和PxPAPb，它们编码可能的PAP。PxPAPa长1149 bp，编码一个382个残基的开放阅读框（ORF），带有一个预测的17个残基的信号肽、一个剪切结构域和一个胰蛋白酶样丝氨酸蛋白酶结构域。PxPAPb长1650 bp，编码一个440个残基的ORF，带有一个预测的20个残基的信号肽、两个剪切结构域和一个胰蛋白酶样丝氨酸蛋白酶结构域。PxPAPa和PxPAPb分别与烟草天蛾（Manduca sexta (L.)）的PAP1和PAP3具有高度的序列相似性。我们还使用实时定量聚合酶链反应检测了PxPAPa、PxPAPb和另一个剪切结构域丝氨酸蛋白酶基因pxPAP3对不同微生物刺激的转录模式。结果表明，藤黄微球菌和大肠杆菌可显著提高PxPAPa的转录丰度，但白色念珠菌不能。PxPAPb仅由藤黄微球菌诱导，而pxPAP3可被测试中的所有微生物诱导，但藤黄微球菌、大肠杆菌和白色念珠菌的转录模式完全不同。这项研究为免疫反应过程中发生的分子事件提供了新的见解，特别是通过宿主昆虫血细胞参与病原体或寄生虫入侵者的包囊化和结节形成的黑化级联反应。
