家蚕几丁质去乙酰化酶BmCDA2的基因表达和免疫定位

Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm

摘要:

几丁质的去乙酰化修饰与昆虫的发育变态密切相关，几丁质去乙酰化酶(chitin deacetylase, CDA)是这个过程中的关键酶。家蚕(Bombyx mori)是鳞翅目昆虫的代表性昆虫，目前对家蚕CDAs的研究较少。为了更好地揭示BmCDAs对家蚕变态发育的作用，本研究采用生物信息学分析、蛋白表达纯化以及免疫荧光定位等方法对表皮中高量表达的BmCDA2进行了研究。结果发现，BmCDA2有两种mRNA拼接形式BmCDA2a和BmCDA2b，分别在幼虫眠期和化蛹期表皮高量表达，两个基因均有几丁质去乙酰化酶催化结构域(catalytic domain)、几丁质结合结构域(chitin binding domain)和低密度脂蛋白受体结构域(low density lipoprotein receptor domain)；Western blotting结果显示，该蛋白在表皮存在，荧光免疫定位发现BmCDA2蛋白随着幼虫新表皮的生成而逐渐增多，推测BmCDA2可能参与了幼虫新表皮的形成。该结果丰富了家蚕CDAs的生物学功能信息，也为其他昆虫CDA的研究提供一些有价值的参考。

Abstract:

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.