甜菜夜蛾SeCDA8的基因克隆、蛋白表达及几丁质结合活性分析

几丁质脱乙酰酶（chitin deacetylase，CDA）能催化几丁质脱乙酰化产生壳聚糖，在几丁质降解过程中发挥着重要作用。本研究通过分析甜菜夜蛾（Spodoptera exigua）转录组得到编码甜菜夜蛾几丁质脱乙酰酶的全长CDS序列，命名为SeCDA8（GenBank登录号为OL689577），其开放阅读框为1158 bp，编码385个氨基酸。NCBI Blast和系统进化分析表明其属于肠道特异CDA蛋白，与斜纹夜蛾CDA相似度最高。成功构建原核重组表达载体pET30a-SeCDA8，转化大肠杆菌，经超声破碎后在沉淀中表达45 kD的重组蛋白。重组SeCDA8蛋白的几丁质结合活性分析结果显示，SeCDA8蛋白与再生几丁质的结合活性较高于胶体几丁质，但均较弱。qRT-PCR分析显示，SeCDA8在前肠和中肠前部高表达，推测其可能与围食膜形成有关。通过探究甜菜夜蛾几丁质脱乙酰酶SeCDA8蛋白的几丁质结合活性和组织定位，为更加深入地探究第Ⅴ类CDA的生理功能提供了重要的理论依据。

Gene Cloning,Protein Expression and Chitin Binding Analysis of SeCDA8 from Beet armyworm,Spodoptera exigua

Chitin deacetylase (CDA) catalyzes the deacetylation of chitin to chitosan, which plays an important role in the degradation of chitin. A full-length coding sequence (CDS) of SeCDA8 was obtained from the transcripome of Spodoptera exigua, named as SeCDA8 (GenBank number of OL689577) with an ORF of 1158 bp encoding 385 amino acid residues. Alignment of the sequence of SeCDA8 showed the highest sequence similarity to the sequence of CDA8 from Spodoptera litura by NCBI Blast and phylogenetic analysis. The prokaryotic recombinant expression vector pET30a-SeCDA8 was successfully constructed and transformed into E. coli Rosetta. The molecular weight of 45 kD recombinant protein was expressed in the precipitation after ultrasonic fragmentation. The chitin-binding activity of SeCDA8 protein to the regenerated chitin was higher than colloidal chitin. Both of them were weak. qRT-PCR analysis of SeCDA8 of midgut tissues from different regions showed that the SeCDA8 was more abundant both in the most anterior region of the midgut and the foregut, indicating that SeCDA8 may be related to the initial formation of peritrophic membrane. The results in this study provide an important theoretical basis for further exploring the physiological function of Group V CDA.