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【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Eo AQP1(Gen Bank登录号:KT819587),Eo AQP1 c DNA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,Eo AQP1在鳞翅目昆虫中高度保守。跨膜结构和水分渗透模拟表明,Eo AQP1具有经典的水分渗透模型。qRT-PCR结果表明,Eo AQP1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,Eo AQP1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,Eo AQP1多克隆抗体特异性较好,可用于后续相关实验。【结论】获得了Eo AQP1的c DNA序列,明确了Eo AQP1的生物学特征,阐明了Eo AQP1的时空表达特性,成功制备了Eo AQP1多克隆抗体,初步了解了Eo AQP1的亚细胞定位,为进一步研究Eo AQP1的水分渗透机理奠定了分子基础。
【Aim】 Aquaporin (AQP) is a transmembrane transporter widely found in mammals, plants and microorganisms. It plays an important role in cellular water transport, ion-selective permeability and osmotic pressure balance Important role. The purpose of this study was to analyze the genetic characteristics of aquaporin AQP1 in Ectropis obliqua Prout and to understand its subcellular localization based on the preparation of polyclonal antibodies. 【Method】 The full-length cDNA of AQP1 was cloned by homologous cloning and RACE. The biological information of AQP1 was analyzed by bioinformatics website and software. The quantitative analysis of AQP1 by qRT-PCR The relative expression of AQP1 in different developmental stages and different tissues of 6th instar larvae was studied. The polyclonal antibody of AQP1 was purified by nickel column purification and immunization of New Zealand rabbits. The AQP1 was detected by fluorescence microscopy in the black belly Drosophila melanogaster Drosophila embryos S2 localization distribution, and the use of polyclonal antibodies Western blot. 【Result】 The full length of AQP1 was cloned and identified as Eo AQP1 (Gen Bank accession number: KT819587). The full length of Eo AQP1 cDNA was 1826 bp in length and contained a 780 bp open reading frame encoding a protein of 259 amino acids. Phylogenetic tree and amino acid sequence alignment showed that Eo AQP1 was highly conserved in Lepidoptera. Transmembrane structure and water infiltration simulation show that Eo AQP1 has a classic water infiltration model. The results of qRT-PCR showed that Eo AQP1 was expressed in different tissues at different developmental stages and in 6th instar larvae, and the difference was significant. The results of subcellular localization showed that Eo AQP1 clustered in the periphery of the cell membrane in a round granule but not in the cell membrane, nuclear membrane and cytoplasm. The result of polyclonal antibody Western blot showed that the polyclonal antibody of Eo AQP1 has better specificity and can be used in subsequent related experiments. 【Conclusion】 The cDNA sequence of Eo AQP1 was obtained, the biological characteristics of Eo AQP1 were clarified, the temporal and spatial expression characteristics of Eo AQP1 were elucidated, and the polyclonal antibody of Eo AQP1 was successfully prepared. The subcellular localization of Eo AQP1 was Further study on the mechanism of water infiltration in Eo AQP1 laid the molecular foundation.