hnRNPA1基因在家蚕翅原基组织中的表达与定位分析  

Study on expression and localization of hnRNPA1 in the wing disc of silkworm,Bombyx mori

作　　者：张康 冯启理[1] 邓惠敏[1] ZHANG Kang;FENG Qi-Li;DENG Hui-Min(School of Life Sciences,South China Normal University,SCNU-Institute of Insect Science and Technology,Guangzhou Key Laboratory of Development Regulation and Application Research of Insects,Guangzhou 510006,China)

机构地区：[1]华南师范大学生命科学学院华南师范大学昆虫科学与应用研究所广州市昆虫发育调控与应用重点实验室,广州510631

出　　处：《环境昆虫学报》2019年第2期343-351,共9页Journal of Environmental Entomology

基　　金：国家自然科学基金重点项目(31330071);广东省自然科学杰出青年基金(2017A030306003)

摘　　要：核不均一蛋白A1(hnRNPA1)是一个重要的RNA结合蛋白。本研究旨在获得家蚕hnRNPA1基因的cDNA,并对其在家蚕翅原基组织进行表达和定位分析。以家蚕幼虫期翅原基mRNA为模板通过反转录克隆家蚕BmhnRNPA1基因的全长cDNA,并对其进行生物信息学分析。构建pET32a-BmhnRNPA1蛋白表达载体,表达且纯化得到BmhnRNPA1重组蛋白,并制备该蛋白多克隆抗体,通过实时荧光定量RT-PCR、Western blot和免疫组化方法检测BmhnRNPA1在家蚕幼虫和蛹翅原基组织中的表达与定位。克隆得到了家蚕hnRNPA1基因的全长cDNA片段,其开放阅读框(ORF)序列为951 bp,编码316个氨基酸,预测分子量为34.98 kDa,等电点为5.15。编码蛋白在第18～90个氨基酸和109～181个氨基酸处为保守的RRM结构域。系统进化分析显示,家蚕hnRNPA1与小菜蛾hnRNPA1的亲缘关系最近。QRT-PCR结果显示,BmhnRNPA1在家蚕幼虫和蛹期的翅原基组织中均有表达,且在蛹期第3天的表达量达到峰值。Western blot进一步证实了实验结果。免疫组化分析结果显示,该蛋白存在于翅原基组织中,并定位于细胞核内。家蚕BmhRNPA1具有两个RNA结合结构域,属于hnRNPs家族,定位于细胞核内,表明其可能参与mRNA的选择性剪接作用。本研究结果为进一步探索该基因的功能提供了基础。hnRNPA1 (heterogeneous protein A1) is an important RNA binding protein. This study is to obtain full length cDNA of hnRNPA1 and to analyze its expression and localization in the wing discs of Bombyx mori . BmhnRNPA1 cDNA was cloned by reverse-transcription PCR (RT-PCR) using mRNA that was isolated from the larvae and its sequence was analyzed. The protein expression vector of pET32a-BmhnRNPA1 was constructed and the recombinant protein was expressed and purified. Polyclonal antibody was generated. The quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry methods were used to analyze the expression and localization of BmhnRNPA1 in the wing discs during the larvae and pupa. The open reading frame of the hnRNPA1 cDNA was 951 bp in length and encoded 316 amino acids. The predicted molecular mass of the protein was 34.98 kDa and the isoelectric point was 5.15. The protein contains two conserved RRM domains between 18 and 90 amino acids and between 109 and 181 amino acids. Phylogenetic analysis showed that the BmhnRNPA1 was closely related to Plutella xylostella hnRNPA1. The results of QRT-PCR showed that BmhnRNPA1 expressed in the wing discs during the larval and pupal stage, and the expression level reached the peak on the 3 rd day of the pupal stage. The results of western blot confirmed the above results. Immunohistochemistry analysis showed that hnRNPA1 expressed in the wing disc and the protein was localized in the nuclei of wing disc cell. This study revealed that BmhRNPA1 has two RNA binding domains, belonging to the hnRNPs family and probably involving in the splicing of premature mRNA. This study provides a foundation for the function analysis of this protein.

关 键 词：家蚕 hnRNPA1 基因表达 免疫组化 RNA剪接 

分 类 号：Q963[生物学—昆虫学] S881.2[农业科学—特种经济动物饲养]