| 华北大黑鳃金龟几丁质脱乙酰酶HoCDA5的分离及定位分析 |
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| 引用本文： | 郜玉杰,赵丹,郭巍,王伟,张雅昆.华北大黑鳃金龟几丁质脱乙酰酶HoCDA5的分离及定位分析[J].河北农业大学学报,2017,40(3). |
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| 作者姓名： | 郜玉杰 赵丹 郭巍 王伟 张雅昆 |
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| 作者单位： | 1. 河北农业大学生命科学学院,河北保定,071000;2. 河北农业大学生命科学学院,河北保定071000;北京农学院植物科学技术学院,北京102206 |
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| 基金项目： | 国家自然科学基金项目，国家现代农业(花生)产业技术体系项目，河北省自然科学基金 |
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| 摘 要： | 为了获得华北大黑鳃金龟(Holotrichia oblita)幼虫几丁质脱乙酰酶5(HoCDA5)及其在不同组织中的表达情况,本研究利用RACE-PCR方法扩增获得编码华北大黑鳃金龟几丁质脱乙酰酶的cDNA基因hocda5(GenBank登录号:ANI87833),该基因的开放阅读框为1140bp,编码379个氨基酸,属于第V类CDA蛋白。在大肠杆菌BL21(DE3)细胞中成功表达约42kDa的蛋白,制备了HoCDA5蛋白的多克隆抗体并对其进行效价测定。利用western blot方法分析了HoCDA5蛋白在不同组织,不同发育时期的表达情况。结果表明:该蛋白主要分布于围食膜,中肠,并且在中肠的前部含量最高,在幼虫的粪便中也检测到了较大量的HoCDA5蛋白,推测华北大黑鳃金龟幼虫的围食膜为I型,HoCDA5可能随着围食膜的新陈代谢排出体外。本研究成功克隆和表达了HoCDA5重组蛋白,制备了多克隆抗体,对该蛋白进行了组织定位分析,为进一步明确HoCDA5蛋白的功能提供了理论依据。
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| 关 键 词： | 华北大黑鳃金龟 几丁质脱乙酰酶 HoCDA5 组织定位分析 |
Isolation and localization analysis of chitin deacetylase (HoCDA5) from Holotrichia oblita |
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| GAO Yu-jie,ZHAO Dan,GUO Wei,WANG Wei,ZHANG Ya-kun.Isolation and localization analysis of chitin deacetylase (HoCDA5) from Holotrichia oblita[J].Journal of Agricultural University of Hebei,2017,40(3). |
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| Authors: | GAO Yu-jie ZHAO Dan GUO Wei WANG Wei ZHANG Ya-kun |
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| Abstract: | To study the expression and localization of HoCDA5 protein in different tissues from the larvae of Holotrichia oblita,hocda5 gene was isolated by rapid amplification of cDNA ends PCR(RACE-PCR).The ORF of hocda5 gene encoded chitin deacetylase was 1140 bp in length (GenBank:ANI87833).The deduced protein sequence showed that HoCDA5 was synthesized as a preprotein of 379 amino acid residues with the predicted molecular weight of 42 kDa,containning the only chitin deacetylase domain and belonging to the group V CDA.The hocda5 was successfully expressed in prokaryotic cells BL21 (DE3).The recombinant protein of HoC-DA5 was used to generate specific polyclonal antiserum.The titer of polyclonal antiserum of HoCDA5 protein was identified.The expression of HoCDA5 protein in different tissues and development stages was detected by western blot.The results showed that HoCDA5 was detected in the midgut and PM,as well as in fecal matter,and it was more abundant in the anterior of the midgut,which indicated that HoCDA5 is involved in updating the PM.In this study,we successfully prepared the polyclonal antibodies of HoCDA5,and analyzed the localization of HoCDA5 from H.oblita,which provided theoretical basis for further investigation of the function of HoCDA5 protein. |
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| Keywords: | Holotrichia oblita chitin deacetylase HoCDA5 localization analysis |
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