小鼠角蛋白10(K10)基因启动子的克隆及活性分析

Clone and Activity Analysis of Keratin 10(K10) Promoter in Mouse

【目的】角蛋白10(K10)是黑色素细胞中黑素体向周围角化细胞迁移的分子标记之一,在研究基因在黑色素细胞与角化细胞相互作用的功能时,可以作为特异的启动子。本研究欲筛选K10较强的启动子片段,为研究K10以及相关基因的功能提供理论依据和奠定理论基础。【方法】从小鼠尾巴提取基因组DNA,经质量鉴定后,采用PCR法扩增K10的6个不同片段(F1—F6),并将其分别亚克隆到p MD18-T载体,经测序验证是否正确;将K10的6个亚克隆片段再克隆到p GL0载体中,产生p GL0-F1—F6构建,用脂质体法转染293T细胞,转染结束后将细胞裂解,并通过双荧光素酶报告基因检测6个片段转染细胞后引起的荧光素酶活性变化,以筛选启动效果最好的启动子片段;用筛选到的K10启动子片段作为特异启动子,替换p GL0载体上的CMV强启动子,并与周期素依赖蛋白激酶5(CDK5)基因进行重组,形成p GL0-F-CDK5构建,用脂质体法转染小鼠皮肤角化细胞,待转染结束后,分别进行细胞爬片、细胞裂解和细胞总RNA的提取,之后用免疫荧光化学、双荧光素酶报告基因检测法和实时荧光定量PCR法检测CDK5的表达定位、表达水平及荧光素酶活性,以检测其在角化细胞中的启动效果;用生物信息法Promoter Scan分析所得到的活性最强的K10启动子片段,发现其可能的转录因子结合位点。【结果】PCR扩增、克隆得到K10启动子的6个片段(F1—F6),片段大小分别为1 201、908、664、787、790、656 bp;质粒p GL0-F1—F6分别转染293T细胞后,通过双荧光报告检测发现长度为787bp的F4启动子活性最强;但F1—F6启动子的活性均弱于p GL0-basic中CMV的启动活性;F4序列中含有基本启动子保守区域的共同序列即TATAAAA,经Promoter Scan分析发现F4序列中含有C/EBPβ、GATA、HSF、CAP等多个转录因子的结合位点,这些位点利于K10在角化细胞中表达;p GL0-F4-CDK5转染角化细胞后,通过荧光蛋白的表达检测载体上GFP报告基因的表达,发现p GL0-F4-CDK5转染角化细胞后引起GFP的表达量明显强于p GL0-basic-CDK5转染组;同时用荧光素酶活性检测p GL0-F4-CDK5在角化细胞中的启动效果,结果发现p GL0-F4-CDK5转染组的荧光比值明显高于对照组,差异显著(P<0.01);经实时荧光定量PCR检测CDK5的表达变化,结果发现p GL0-F4-CDK5转染角化细胞后引起CDK5 m RNA表达量明显高于对照组,差异呈极显著(P<0.01)。上述结果说明F4具有较强的启动子活性,是K10启动子的核心区。【结论】成功筛选了K10的核心启动子区域F4,在角化细胞里具有启动目标基因CDK5表达的功能,因此,F4可作为黑色素细胞与角化细胞相互作用过程中基因功能研究的特异性启动子,为研究K10基因功能提供理论依据。

【Objective】 Keratin 10（K10） is one of the molecular markers of melanosome transfer from melanocytes to the around keratinocytes, which can be used as the specific promoter for the research on the interactions between melanocytes and keratinocytes. Here, the strongest promoter of K10 will be screened to supply the evidence and basis for the research of the function of K10 and other relative genes. 【Method】 Genomic DNA was extracted from a mouse tail. Six fragments of K10 were amplified and subcloned into p MD18-T vector and sequenced. They were then cloned into p GL0 vector to construct p GL0-F1-F6, was transfected into 293 T cells by liposome. The luciferase activity were used to identify the strongest promoter from the 6 fragments of K10 promoter（F1-F6）, which was measured by luciferase reporter（GFP） from the cell lysis and the prediction of transcript factor binding sites by bioinformatics of the sequence. After the strongest promoter of K10 was identified, it was used as a specific promoter, which recombined with p GL0（without CMV） to construct p GL0-F-CDK5. p GL0-F-CDK5 was transfected into keratinocytes of mouse by liposome and then Cyclin-dependent Kinase 5（CDK5） expression was measured by Immunofluorescence chemistry, luciferase reporter（GFP） and quantitative real-time PCR from the cells, cell lysis and total RNA, respectively. 【Result】 Six fragments（F1-F6） of K10 were amplified, cloned and sequenced with the size of 1 201, 908, 664, 787, 790 and 656 bp, respectively. The luciferase activity of F4 with the size of 787 bp was the strongest as measured by the luciferase reporter gene in 293 T cell, which suggested that F4 would be used as a specific promoter. But the luciferase activity of F1-F6 was weaker than that of CMV. In the F4 sequence, the basic conserved region（TATAAAA） and the transcript factor binding sites such as C/EBPβ, GATA, HSF and CAP were found by Promoter Scan software, which promotes the K10 expression in keratinocytes. After transfection, the result of luciferase activity measurement showed that p GL0-F4-CDK5 made GFP expression stronger than p GL0-basic-CDK5 with a significant difference（P〈0.01）, and made CDK5 m RNA expression higher than p GL0-basic-CDK5 with a significant difference（P〈0.01）, which suggested that F4 was the corn region of K10 promoter with the strongest activity. 【Conclusion】 F4 was identified as the corn region of K10 promoter to have the strongest activity in promoting CDK5 expression in keratinocyte, which suggested that F4 would be used as a specific promoter for the gene function research during the interaction between melanocyte and keratinocyte.