暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析

doi:10.16409/j.cnki.2095-039x.2018.05.006

中国生物防治学报 ›› 2018, Vol. 34 ›› Issue (5): 686-692.DOI: 10.16409/j.cnki.2095-039x.2018.05.006

暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析

赵丹¹, 闫晓平¹, 陆秀君¹, 郭巍^1,2

1. 河北农业大学植物保护学院, 保定 071000;
2. 北京农学院植物科学技术学院, 北京 102206

收稿日期:2018-05-18 出版日期:2018-10-08 发布日期:2018-10-15
通讯作者: 郭巍,教授,E-mail:guowei@hebau.edu.cn。
作者简介:赵丹,助理研究员,E-mail:zhaodan@hebau.edu.cn
基金资助:
河北省自然科学基金（C2016204082）；国家花生产业技术体系（CARS-13）

Expression and Characteration of the Chitin Deacetylase 5 Gene from Holotrichia parallela Larvae

ZHAO Dan¹, YAN Xiaoping¹, LU Xiujun¹, GUO Wei^1,2

1. College of Plant Protection, Agricultural University of Hebei, Baoding 071001, China;
2. Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China

Received:2018-05-18 Online:2018-10-08 Published:2018-10-15

摘要/Abstract

摘要： cDNA文库免疫筛选到编码暗黑鳃金龟幼虫几丁质脱乙酰酶HpCDA5基因，序列分析表明HpCDA5含有1个几丁质脱乙酰酶结构域，属于Group V类CDA蛋白。构建重组杆状病毒表达载体pFastBac-HpCDA5，转染昆虫细胞sf9，Western blot分析表明HpCDA5在昆虫细胞sf9中成功表达42 kDa的蛋白。利用qRT-PCR方法分析HpCDA5基因组织表达，结果显示HpCDA5基因在中肠中表达最高，为中肠特异表达蛋白。几丁质结合活性表明HpCDA5蛋白只能被强洗脱剂洗脱，具有很强的几丁质结合活性。本研究通过对暗黑鳃金龟几丁质脱乙酰酶HpCDA5的生化特性研究，为进一步明确HpCDA5的生理功能提供理论依据，并为以HpCDA5蛋白为靶标的暗黑鳃金龟生物防治提供支撑。

关键词: 暗黑鳃金龟, HpCDA5, 细胞表达, 组织表达分析, 几丁质结合活性

Abstract: The HpCDA5 gene encoding chitin deacetylase was obtained by immune-screening the cDNA library of Holotrichia parallela with an anti-PM proteins polyclonal antiserum. The HpCDA5 protein has a polysaccharide deacetylase-like domain, belonging to Group V CDA. The HpCDA5 was successfully expressed in insect cells (sf9) as secreted proteins using recombinant baculoviruses (Bac to Bac system). Western blot analysis showed that the apparent molecular weight for recombinant HpCDA5 were about 42 kDa. qRT-PCR approach revealed that HpCDA5 has high expression in midgut. Subsequent chitin binding assays demonstrated that HpCDA5 had chitin binding affinity. The characterization of recombinant HpCDA5 laid a foundation for further studies and physiological functions of the HpCDA5 protein, and provide a theoretical basis for the biocontrol of H. parallela using CDA protein as target.

Key words: Holotrichia parallela, HpCDA5, insect cell expression, tissue expression, chitin binding affinity

中图分类号:

S433.5

赵丹, 闫晓平, 陆秀君, 郭巍. 暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析[J]. 中国生物防治学报, 2018, 34(5): 686-692.

ZHAO Dan, YAN Xiaoping, LU Xiujun, GUO Wei. Expression and Characteration of the Chitin Deacetylase 5 Gene from Holotrichia parallela Larvae[J]. journal1, 2018, 34(5): 686-692.

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暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析

Expression and Characteration of the Chitin Deacetylase 5 Gene from Holotrichia parallela Larvae

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