摘要
旨在研究高硫角蛋白基因启动子及相关调控序列的表达活性。以绵羊基因组为模板,克隆KAP1.3基因启动子序列,对其进行5个系列缺失片段pGL4.10表达载体构建,将不同重组质粒转染小鼠成纤维细胞,通过裂解细胞测定其荧光值方法来检测启动子的表达活性。结果显示,除KAP1.3-P5片段其余片段均具有表达活性,其中KAP1.3-P1片段表达活性最高,而缺失-944至-707处的KAP1.3-P2对启动子的活性没有显著影响,但缺失-944至-394处KAP1.3-P3片段对启动子活性影响比较大,从P3和P4的结果中可以看出,虽然两个片段都缺失了转录因子bHLH的结合位点,但P4的活性比P3高出约20%,由此推测在-394至-195之间可能存在一个负调控元件,在-195和-4之间可能存在一个增强元件,但具体的调控元件还需要再进行细致地分析,优化出活性最高且特异性不变的启动子片段。
The keratin is main structure albumen of the wool, which is the most important part of skin. In order to study the expression activity of high-sulfur keratin gene promoter and associated regulatory sequences, our experiment regarded the genomic DNA from the sheep as the template, through molecular biologic methods to clone the KAP1.3 gene, listing the five missing fra^nents after joint pGIA. 10 vector, then detecting the promoter activity through the method of transfecting mouse fibroblasts by cationic liposome. The results showed that all the fragments had expression activity except KAP1.3-P5, and KAP1.3-P1 expressed the highest activity, while KAP1.3-P2 lacked the length -944 to -707 did not significantly affect promoter activity, it can be seen from the results of the P3 and P4, the two fragments are missing transcription factor bHLH binding sites, but the P4 activity higher than the P3 by about 20%, which indicated that between -394 and -195 maybe presence of a negative regulatory element and between -195 and -4, there maybe an enhanced element, the specific regulatory elements needs the meticulous anal2csis, also the activity and specificity of constant promoter fragment.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第9期74-79,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(31001002
31160460)
国家"863"计划项目(2011AA100307)
国家科技支撑计划(2011BAD28B05-1-1)
转基因生物新品种培育重大专项(2008ZX08008-001
2009ZX08008-001B)
国家绒毛用羊产业技术体系(CARS-40-07)
新疆兵团博士基金项目(2010JC10)
新疆兵团育种攻关项目(2011BA006)
