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shRNA下调家蚕细胞系BmCREB的表达
樊宗标,张鸿雁,赵怡姣,宋红生
0
(上海大学生命科学学院,上海 200444)
摘要:
shRNA(short hairpin RNA)是一种干扰基因表达、研究基因功能的有效方法。CREB(cAMP response element binding protein)是真核生物重要而又保守的转录调控因子,参与许多生理功能的调节。作者前期的研究发现,家蚕CREB (BmCREB) 的表达与家蚕滞育的诱导密切相关。为进一步阐明BmCREB调控家蚕滞育的分子机制,利用pMD18-T质粒构建BmCREB的干扰质粒pMD18-U6-shRNA,转染家蚕卵巢细胞系 (BmN),对其下调BmCREB的表达进行研究。结果表明,在BmN细胞系中转染1 μg shRNA,72 h后能有效地下调BmCREB蛋白的表达量,最高干扰效率达63.0%。不同干扰片段shRNA质粒的干扰效果差异很大,其中T-227的干扰效果最好。这些结果为进一步利用shRNA方法在家蚕体内下调基因表达,研究BmCREB基因与家蚕滞育的关系奠定基础。
关键词:  shRNA  家蚕  BmCREB
DOI:10.13610/j.cnki.1672-352x.20150625.006
投稿时间:2015-04-27
基金项目:国家自然科学基金面上项目(31172147)和上海市教委创新项目(09YZ38)共同资助。
Down-regulation of BmCREB by shRNA in Bombyx mori cell lines
FAN Zongbiao,ZHANG Hongyan,ZHAO Yijiao,SONG Hongsheng
(College of Life Science, Shanghai University, Shanghai 200444)
Abstract:
Short hairpin RNA (shRNA) is an effective method for interfering gene expression and gene function research. CREB (cAMP response element binding protein), an important and conservative transcriptional factor in eukaryote, regulates many physiological functions. According to our previous research, Bombyx mori CREB (BmCREB) is closely relative to the diapause induction in the silkworm. In order to clarify the molecular mechanism of BmCREB in diapause induction, we constructed the interfering vector pMD18-U6-shRNA of BmCREB shRNA and transfected in a Bombyx mori ovary cell line (BmN). The expression of BmCREB was detected by Western blot and RT-PCR. The results showed that BmCREB was efficiently reduced by 63.0% with the transfection of 1 μg T-227 plasmid in 72 hours. The down-regulation of BmCREB was significant within different shRNA plasmids. Plasmid T-227 had the best efficiency. These results would lay a foundation for the further research on BmCREB mechanism of the Bombyx mori diapause by shRNA in vivo.
Key words:  shRNA  Bombyx mori  BmCREB

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