昆虫几丁质脱乙酰基酶BmCDA1和OfCDA1的性质研究-生物化工专业论文.docxVIP

研究生优秀毕业论文 大连理工大学硕士学位论文摘 大连理工大学硕士学位论文 摘 要 几丁质脱乙酰基酶属于糖酯酶4家族，能催化几丁质的乙酰氨基水解生成壳聚糖。 昆虫几丁质脱乙酰基酶1(CDAl)主要在昆虫蜕皮过程的表皮中表达，很可能参与新 表皮中几丁质的装配过程，因此具有重要的生理功能。本文以鳞翅目模式生物家蚕的 BmCDAl和农业害虫亚洲玉米螟的OfCDAl为研究对象，主要取得以下研究结果： 1．利用PCR分别调取了家蚕和亚洲玉米螟中的BmCDAl和OfCDAl基因。 BmCDAl和OfCDAl的开放阅读框分别为1620bp和1617bp。进化分析表明，BmCDAl 和OfCDAl同属于昆虫Group I几丁质脱乙酰基酶，序列一致性高达96％，说明鳞翅目 昆虫CDAl可能具有很高的保守性。与真菌及细菌来源的几丁质脱乙酰基酶一样，序列 分析表明两者含有5个与催化相关的保守基序。 2．BmCDAl的基因转录水平研究表明，在不同组织中，Bm∞爿J在表皮中表达量 最高；在不同发育过程中，BmCDAl在蛹期表达量明显升高。说明该酶主要存在于表皮 中并且在化蛹时发挥重要功能。注射蜕皮激素12 h后BmCDAJ表达量发生上调，说明 该基因的表达受蜕皮激素的调控。 3．选择不同的表达系统对不同来源的CDA进行重组表达。在大肠杆菌中， BmCDAl以包涵体形式表达，未能实现分离纯化；在毕赤酵母中，BmCDAl和OfCDAl 能够表达，甲醇诱导144h后，可以通过硫酸铵沉淀和金属螯合层析两步从发酵液上清 中纯化获得重组蛋白。 4．采用荧光胺法和3．甲基一2．苯并噻唑酮腙法对重组表达的CDA活性进行检测，结 果表明：BmCDAl没有检测出酶活性。但几丁质结合实验发现，BmCDAl具有结合胶 体几丁质的能力。 关键词：几丁质；几丁质脱乙酰基酶；重组表达；家蚕；亚洲玉米螟 万方数据 昆虫几丁质脱乙酰基酶BmCDAl和OfCDAl的性质研究B 昆虫几丁质脱乙酰基酶BmCDAl和OfCDAl的性质研究 B iochemical characterization of the insect chitin deacetylases BmCDA 1 and OfCDAl Abstract ChjtiIl deacetylase(CDA)，a member of carboh)rdrate esterase family 4，catalyzes the hydrolysis of acetylamino of chitin to generate chitosan．Insect chitin deacetylase l(CDA 1)iS mainly expressed in epidermis during molting，perhaps playing a critical role by participating in chitin assembling in the new cuticle．In this thesis，two insect CDA 1 S including BmCDA 1 from Bombyx mori and OfCDAl from Ostriniafurnacalis were studied．The following is the results obtained： 1．The eDNA sequences of C删1 from B．mori and D furnacalis were obtained by using RT．PCR．Both Bm(Z啊j and OfCDA 1 consisted of all open reading frame of 1 620bp and 1 6 1 7bp respectively．Phylogenic analyses indicated that they both belonged to Group I insect CDA and shared an identity of 96％in similarity．Secquence analyses demonstated that both of them contained five conserved motifs，which were known in fungi and bacteria and were deduced to be responsible to catalysis． 2．The tissue．and development—specific expression pattem indicated that BmC删J『 was mainly expressed in epidermis an