茶尺蠖水通道蛋白EoAQP1的cDNA克隆、多克隆抗体制备及亚细胞定位

doi:10.16380/j.kcxb.2016.04.003

摘要/Abstract

摘要： 【目的】水通道蛋白（aquaporin, AQP）是一种广泛存在于哺乳动物、植物和微生物中的跨膜转运蛋白，它在细胞水分运输、离子选择透过性和渗透压平衡等过程中发挥着重要的作用。本研究旨在分析茶尺蠖Ectropis obliqua Prout水通道蛋白AQP1的基因特性，在制备多克隆抗体的基础上了解其亚细胞定位分布。【方法】采用同源克隆方法并结合RACE技术克隆茶尺蠖AQP1的基因全长，通过生物信息学网站和软件分析茶尺蠖AQP1的生物学信息；通过实时荧光定量PCR技术（qRT-PCR）检测茶尺蠖AQP1在不同发育阶段和6龄幼虫不同组织中的相对表达量；通过原核表达、镍柱纯化并免疫新西兰兔制备了茶尺蠖AQP1的多克隆抗体；通过荧光显微镜观测了茶尺蠖AQP1在黑腹果蝇Drosophila melanogaster胚胎细胞S2中的定位分布，并利用多克隆抗体进行了Western blot验证。【结果】克隆并鉴定了茶尺蠖AQP1全长，将其命名为EoAQP1（GenBank登录号: KT819587），EoAQP1 cDNA全长1 826 bp，含有780 bp开放阅读框，编码259个氨基酸。系统进化树和氨基酸序列同源性比对表明，EoAQP1在鳞翅目昆虫中高度保守。跨膜结构和水分渗透模拟表明，EoAQP1具有经典的水分渗透模型。qRT-PCR结果表明，EoAQP1在不同发育时期和6龄幼虫不同组织中均有表达且差异显著。亚细胞定位结果表明，EoAQP1以圆形颗粒状成群聚集于细胞膜周边，而在细胞膜、核膜和细胞质等处均不表达。多克隆抗体Western blot检测结果表明，EoAQP1多克隆抗体特异性较好，可用于后续相关实验。【结论】获得了EoAQP1的cDNA序列，明确了EoAQP1的生物学特征，阐明了EoAQP1的时空表达特性，成功制备了EoAQP1多克隆抗体，初步了解了EoAQP1的亚细胞定位，为进一步研究EoAQP1的水分渗透机理奠定了分子基础。

关键词: 茶尺蠖, 水通道蛋白, 克隆, 生物信息学, 表达模式, 亚细胞定位, 多克隆抗体

Abstract: 【Aim】 Aquaporin (AQP) is one of the transmembrane proteins widely existing in mammals, plants, and microorganisms, and plays an important role in the processes of cell moisture transportation, selective ion permeability and osmotic balance. This study aims to understand the gene characteristics of AQP1 from Ectropis obliqua Prout, and its subcellular localization based on polyclonal antibody preparation.【Methods】 The complete cDNA of AQP1 was cloned from E. obliqua by RT-PCR and RACE technologies. The bioinformatics analysis was carried out by online website and edited on the bioinformatics software. The mRNA relative expression levels in different developmental stages and different tissues of the 6th instar larvae of E. obliqua were investigated by real-time quantitative PCR (qRT-PCR). The polyclonal antibody was prepared by immunizing a male New Zealand white rabbit after prokaryotic expression and Ni-sepharose purification. Subcellular localization in Drosophila melanogaster embryonic cells (S2 cells) was observed by fluorescence microscope, and Western blot analysis was performed with the polyclonal antibody of AQP1 from E. obliqua. 【Results】 The AQP1 cDNA was cloned successfully from E. obliqua and named EoAQP1 (GenBank accession no.: KT819587). It is 1 826 bp in length, including a 780 bp of open reading frame (ORF), which encodes 259 amino acids. Phylogenetic tree analysis and alignment of amino acid sequences showed that EoAQP1 is highly conserved with AQP1 proteins of other species of Lepidoptera. Transmembrane structure and water percolating simulation showed that EoAQP1 has a classical water percolation model. The qRT-PCR result revealed that EoAQP1 was expressed in all developmental stages and various tissues of the 6th instar larvae of E. obliqua at different mRNA expression levels. Subcellular localization showed that EoAQP1 aggregated around cell membrane with circular and granular models, but was not expressed in cell membrane, cytoplasm or nuclear membrane. Western blot analysis demonstrated that the obtained polyclonal antibody had high specificity and could be used for further experiments. 【Conclusion】 The nucleotide sequence, bioinformatics and expression profiling of aquaporin 1 gene EoAQP1 in E. obliqua were clarified. With the obtained polyclonal antibody, its subcellular localization of EoAQP1 was preliminarily investigated. These results provide a foundation for further research on the mechanism of water infiltration of EoAQP1.

Key words: Ectropis obliqua, aquaporin, cloning, bioinformatics, expression patterns, subcellular localization, polyclonal antibody

李良德, 王定锋, 刘丰静, 李慧玲, 张辉, 吴光远. 茶尺蠖水通道蛋白EoAQP1的cDNA克隆、多克隆抗体制备及亚细胞定位[J]. 昆虫学报, 2016, 59(4): 382-391.

LI Liang-De, WANG Ding-Feng, LIU Feng-Jing, LI Hui-Ling, ZHANG Hui, WU Guang-Yuan. cDNA cloning, preparation of polyclonal antibody and subcellular localization of aquaporin 1 (AQP1) in Ectropis obliqua (Lepidoptera: Geoqmetridae)[J]. Acta Entomologica Sinica, 2016, 59(4): 382-391.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://www.insect.org.cn/CN/10.16380/j.kcxb.2016.04.003

http://www.insect.org.cn/CN/Y2016/V59/I4/382

[1]	张海燕, 吴金鑫, 张云贵, 赵萍, 林英. 家蚕非滞育红卵突变体Re-nd突变基因的定位克隆[J]. 昆虫学报, 2022, 65(6): 668-674.
[2]	李亚子, 赵丹, 郭晓昌, 吴涵, 刘兆瑞, 郭巍. 甜菜夜蛾肽聚糖识别蛋白基因SePGRP-SA的克隆及功能鉴定[J]. 昆虫学报, 2022, 65(4): 417-426.
[3]	尹恒, 傅子卓, 杨晓霞, 袁东雪, 毛新芳, 刘忠渊. 茶尺蠖无机焦磷酸酶EoPPase672的表达与功能研究[J]. 昆虫学报, 2022, 65(4): 427-436.
[4]	王雅丽, 赵瑞, 王美, 赵萌萌, 张晓晨, 李锐. 星豹蛛四个羧酸酯酶基因克隆及溴氰菊酯胁迫下的表达模式[J]. 昆虫学报, 2022, 65(4): 490-499.
[5]	付裕, 王倩, 张勇, 陈巨莲. 荻草谷网蚜唾液蛋白基因Sm13498的克隆及功能分析[J]. 昆虫学报, 2021, 64(9): 1009-1019.
[6]	张肖肖, 雷丹, 李香盈, 陈斌. 中华按蚊含LY结构域的胰蛋白酶基因的分子特征及表达模式分析[J]. 昆虫学报, 2021, 64(9): 1050-1060.
[7]	熊亮, 敖塘堰, 张真, 马振刚, 周泽扬. 东方蜜蜂微孢子虫一新型孢壁蛋白的基因克隆、表达及亚细胞定位[J]. 昆虫学报, 2021, 64(9): 1070-1079.
[8]	王宏民, 张静, 张冲, 庄国动, 郑海霞, 张仙红. 绿豆象气味结合蛋白基因的克隆及表达分析[J]. 昆虫学报, 2021, 64(8): 920-928.
[9]	陈秋应, 杨喜, 游东蕊, 杨沐, 徐志峰, 肖伟. 桃蛀螟性信息素结合蛋白CpunPBP3的表达模式及性信息素结合特性(英文)[J]. 昆虫学报, 2021, 64(3): 318-326.
[10]	郭洪刚, 魏春花, 张民照, 覃晓春, 杜艳丽. 桃蛀螟气味结合蛋白CpunOBP3和CpunOBP4的基因克隆、原核表达及配体结合特性分析[J]. 昆虫学报, 2021, 64(3): 327-336.
[11]	丁朝阳, 赵乐, 刘苏, 李茂业. 菜粉蝶热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应[J]. 昆虫学报, 2021, 64(12): 1407-1416.
[12]	梁玉键, 张涛, 李草, 郅军锐. 草地贪夜蛾海藻糖合成酶基因的克隆、时空表达谱及对温度胁迫的响应[J]. 昆虫学报, 2021, 64(12): 1417-1426.
[13]	李琼艳, 陈安利, 荀利杰, 刘增虎, 廖鹏飞, 杨伟克, 董占鹏. 琥珀蚕丝腺转录因子基因AaSGF-1的克隆、原核表达及组织表达分析[J]. 昆虫学报, 2021, 64(1): 41-50.
[14]	张玉明, 邵梦华, 李亚静, 冯琴, 魏丽亚, 柳峰松. Mdogg1基因参与家蝇体内氧化还原平衡的维持[J]. 昆虫学报, 2021, 64(1): 51-60.
[15]	李游山, 路庆君, 杨玺, 张杰, 罗竹星, 夏庆友, 赵萍. 家蚕抗真菌因子BmSPI39对球孢白僵菌入侵的表达响应[J]. 昆虫学报, 2021, 64(1): 61-69.

茶尺蠖水通道蛋白EoAQP1的cDNA克隆、多克隆抗体制备及亚细胞定位

cDNA cloning, preparation of polyclonal antibody and subcellular localization of aquaporin 1 (AQP1) in Ectropis obliqua (Lepidoptera: Geoqmetridae)

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0