Zhang Huanhuan, Hu Weina, Xiao Miaomiao, Ou Shiyi, Hu Qiongbo
Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of Agriculture, South China Agricultural University , Guangzhou 510642, China.
Department of Food Science and Engineering, Jinan University , Guangzhou 510632, China.
J Agric Food Chem. 2017 Nov 15;65(45):9849-9853. doi: 10.1021/acs.jafc.7b03734. Epub 2017 Nov 2.
Destruxin A (DA) is a cyclodepsipeptidic mycotoxin isolated from the entomopathogenic fungus, Metarhizium anisopliae. It has insecticidal activity against host insect's innate immunity system, but the molecular mechanism is not yet elucidated. In our previous experiment, four HSPs (heat shock proteins, BmHSP70-3, BmHSP75, BmHSP83, and BmHSCP) were characterized from the specific protein electrophoretic bands of Bombyx mori Bm12 cell line treated with DA in the test of drug affinity responsive target stability (DARTS), which implied that these HSPs might be kinds of DA-affinity proteins, or DA induces them up-regulated expression. Therefore, in current research, the interactions of DA and HSPs were explored through analysis of bio-layer interferometry (BLI) employing FortBio OcteteQK. The expression levels of HSPs genes were surveyed by quantitative real-time polymerase chain reaction (qPCR). The results indicated that DA had no interactions with BmHSP70-3, BmHSP75, and BmHSP83, but had affinity to BmHSCP with a K value of 88.1 μM, in BLI analysis. However, the expression levels of all HSPs genes were significantly up-regulated after the Bm12 cells were treated by DA. In conclusion, DA can induce the four HSPs expression in Bm12 cells, but DA only binds to BmHSCP. Our research provides new insights on understanding of the action mechanisms of destruxins.
destruxin A（DA）是一种从昆虫病原真菌绿僵菌中分离出的环缩肽类霉菌毒素。它对宿主昆虫的先天免疫系统具有杀虫活性，但其分子机制尚未阐明。在我们之前的实验中，通过药物亲和力响应靶点稳定性（DARTS）测试，从用DA处理的家蚕Bm12细胞系的特异性蛋白质电泳条带中鉴定出了四种热休克蛋白（HSPs，即BmHSP70-3、BmHSP75、BmHSP83和BmHSCP），这表明这些HSPs可能是DA的亲和蛋白，或者是DA诱导它们上调表达。因此，在当前研究中，我们使用FortBio OcteteQK通过生物层干涉术（BLI）分析来探究DA与HSPs之间的相互作用。通过定量实时聚合酶链反应（qPCR）检测HSPs基因的表达水平。结果表明，在BLI分析中，DA与BmHSP70-3、BmHSP75和BmHSP83没有相互作用，但与BmHSCP有亲和力，K值为88.1 μM。然而，在用DA处理Bm12细胞后，所有HSPs基因的表达水平均显著上调。总之，DA可诱导Bm12细胞中这四种HSPs的表达，但DA仅与BmHSCP结合。我们的研究为理解 destruxins的作用机制提供了新的见解。
