Abstract：
Transglutaminase (TGase) is a key enzyme that mediates hemolymph coagulation and is thought to contribute to the elimination of pathogenic microorganisms in invertebrates. The objective of this study was to elucidate the involvement of TGase in insect immune responses via functional analysis of this enzyme in the oriental armyworm, Mythimna separata, using recombinant proteins and RNA interference technique. We identified two TGase genes, mystgase1 and mystgase2, in Mythimna separata and found that both genes are expressed in all surveyed tissues in M. separata larvae. Significant changes were induced in hemocytes following Escherichia coli injection. Injection of Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Escherichia coli and Serratia marcescens) into larvae triggered a time-specific induction of both mystgase1 and mystgase2 in hemocytes. Recombinant MysTGase1 and MysTGase2 proteins bound to both E. coli and M. luteus, localizing within bacterial clusters and resulting in agglutination in a Ca2+-dependent manner. The hemocytes of larvae injected with recombinant MysTGase1 or MysTGase2 exhibited enhanced phagocytic ability against E. coli, improved in vivo bacterial clearance, and increased resistance to S. marcescens, decreasing larval mortality rate. Conversely, RNA interference targeting mystgase1 or mystgase2 significantly reduced hemocyte phagocytic capability, decreased bacterial clearance, and increased susceptibility to S. marcescens infection, thereby increasing larval mortality rate. The findings of this study are anticipated to expand our understanding of the function of TGases within insect immune responses and may contribute to developing new pest control strategies.
摘要：
转谷氨酰胺酶（TGase）是介导血淋巴凝固的关键酶，被认为有助于消除无脊椎动物中的病原微生物。这项研究的目的是通过对东方粘虫中TGase酶的功能分析来阐明TGase在昆虫免疫反应中的参与，Mythimnaseparata,使用重组蛋白和RNA干扰技术。我们鉴定了两个TGase基因,mystgase1和mystgase2，在Mythimnaseparata中，发现这两个基因在M.separata幼虫的所有调查组织中均表达。大肠杆菌注射后，血细胞发生了显着变化。将革兰氏阳性菌（黄体微球菌）和革兰氏阴性菌（大肠杆菌和粘质沙雷氏菌）注射到幼虫中，会触发血细胞中mystgase1和mystgase2的时间特异性诱导。重组MysTGase1和MysTGase2蛋白与大肠杆菌和黄体分枝杆菌结合，定位在细菌簇内，并以Ca2依赖性方式导致凝集。注射重组MysTGase1或MysTGase2的幼虫血细胞对大肠杆菌的吞噬能力增强，改善体内细菌清除率，并且增加了对粘质链球菌的抗性,降低幼虫死亡率。相反，靶向mystgase1或mystgase2的RNA干扰显着降低血细胞的吞噬能力，细菌清除率下降，和增加对粘质链球菌感染的易感性，从而增加幼虫死亡率。这项研究的结果有望扩大我们对TGases在昆虫免疫反应中的功能的理解，并可能有助于开发新的害虫控制策略。
