家蚕let-7 microRNA靶基因Bmlin-41的克隆表达与调控

周兰庭1,2 周挺1 高君凌1 王伟1 吴小燕1 黄亚玺1 夏庆友1 刘仕平1

(1.西南大学家蚕基因组生物学国家重点实验室, 重庆 400716)
(2.湖北文理学院医学院, 湖北襄阳 441053)

【摘要】异时性基因调控细胞增殖和个体发育阶段的转换。家蚕异时性基因在家蚕变态发育过程中也很可能具有重要的调控作用,但它们的表达模式、生物学功能以及与micro RNA之间的关系却鲜有报道。本研究首先利用果蝇同源基因lin-41搜索家蚕基因组数据库中相似序列,设计引物扩增Bmlin-41的编码序列,克隆了家蚕Bmlin-41基因CDS,其长度为2 166 bp,编码721个氨基酸,含有B-box和NHL结构域;随后,利用RT-PCR、q PCR技术并结合已有的家蚕全基因组芯片数据研究了Bmlin-41在家蚕中的时空表达模式,发现Bmlin-41在从家蚕胚胎到成虫的发育过程中呈逐渐递增的表达趋势,在五龄3 d不同组织中,于卵巢里表达量最高,精巢和中肠次之,而其余组织中低量表达或不表达;最后,利用3′RACE克隆了Bmlin-41基因的3′UTR,全长1 434 bp,用在线软件RNAhybrid预测发现Bmlin-41的3′UTR上存在bmo-let-7靶位点,构建了含Bmlin-41 3′UTR的双荧光素酶报告基因载体,在S2细胞上共转染Bmlin-41 3′UTR和bmo-let-7的模拟物(Mimics)和拮抗剂(Antagomir),bmo-let-7 mimics显著下调Bmlin-41,bmo-let-7 antagomir显著上调Bmlin-41,证实了Bmlin-41是bmo-let-7的靶基因。以上研究结果为深入研究let-7 mi RNA和Bmlin-41的功能,揭示Bmlin-41和bmo-let-7在家蚕变态发育过程中的调控关系提供了新的线索。

【关键词】 家蚕; 异时性基因; 家蚕let-7 miRNA; Bmlin-41; 表达模式; 靶基因;

【DOI】

【基金资助】 国家重点基础研究发展计划(973计划)(No.2012CB114602) National Basic Research Program of China(973 Program)(No.2012CB114602) 国家自然科学基金(Nos.31071136,31571334) National Natural Science Foundation of China(Nos.31071136,1571334) 重庆市基础与前沿研究计划(No.cstc2014jcyjA00025)资助 Fundamental and Advanced Research Projects of Chongqing(No.cstc2014jcyjA00025)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 635-647

May 2016

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摘要

  • 1 材料与方法
  • 2 结果与分析
  • 3 讨论
  • 4 结论
  • 参考文献