基因组学与应用生物学
基因組學與應用生物學
기인조학여응용생물학
GENOMICS AND APPLIED BIOLOGY
2013年
4期
459-466
,共8页
李飞%许厚强%陈伟%陈祥%刘敏%桓聪聪
李飛%許厚彊%陳偉%陳祥%劉敏%桓聰聰
리비%허후강%진위%진상%류민%환총총
MyoDI基因%启动子%荧光素酶%C2C12%3T3-L1
MyoDI基因%啟動子%熒光素酶%C2C12%3T3-L1
MyoDI기인%계동자%형광소매%C2C12%3T3-L1
MyoDIgenes%Promoter%Luciferase%C2C12%3T3-L1
为了克隆关岭牛MyoD玉基因启动子并验证其启动活性，根据GenBank中牛的MyoD玉基因序列设计PCR引物，用PCR技术扩增牛MyoD玉基因的启动子，构建重组克隆载体pUCmT-MyoD玉；并通过PCR扩增、限制性酶切、测序及生物信息学分析对阳性克隆进行鉴定；构建报告质粒pGL3-MyoD玉，并将其转染小鼠C2C12、3T3-L1细胞系，检测其24 h后的双荧光素酶活性。实验结果获得了关岭牛MyoD玉基因启动子，其序列长度为993 bp，并成功构建了MyoD玉启动子报告质粒；双荧光素酶活性检测表明pGL3-MyoD玉在小鼠C2C12细胞中的表达是pGL3空载体40.65倍，在小鼠3T3-L1细胞中的表达是空载体的1.13倍，MyoD玉启动子在小鼠C2C12细胞中的表达高于3T3-L1细胞(**p<0.01)。结果表明关岭牛MyoD玉基因启动子具有启动活性，在小鼠骨骼肌细胞中特异性表达。
為瞭剋隆關嶺牛MyoD玉基因啟動子併驗證其啟動活性，根據GenBank中牛的MyoD玉基因序列設計PCR引物，用PCR技術擴增牛MyoD玉基因的啟動子，構建重組剋隆載體pUCmT-MyoD玉；併通過PCR擴增、限製性酶切、測序及生物信息學分析對暘性剋隆進行鑒定；構建報告質粒pGL3-MyoD玉，併將其轉染小鼠C2C12、3T3-L1細胞繫，檢測其24 h後的雙熒光素酶活性。實驗結果穫得瞭關嶺牛MyoD玉基因啟動子，其序列長度為993 bp，併成功構建瞭MyoD玉啟動子報告質粒；雙熒光素酶活性檢測錶明pGL3-MyoD玉在小鼠C2C12細胞中的錶達是pGL3空載體40.65倍，在小鼠3T3-L1細胞中的錶達是空載體的1.13倍，MyoD玉啟動子在小鼠C2C12細胞中的錶達高于3T3-L1細胞(**p<0.01)。結果錶明關嶺牛MyoD玉基因啟動子具有啟動活性，在小鼠骨骼肌細胞中特異性錶達。
위료극륭관령우MyoD옥기인계동자병험증기계동활성，근거GenBank중우적MyoD옥기인서렬설계PCR인물，용PCR기술확증우MyoD옥기인적계동자，구건중조극륭재체pUCmT-MyoD옥；병통과PCR확증、한제성매절、측서급생물신식학분석대양성극륭진행감정；구건보고질립pGL3-MyoD옥，병장기전염소서C2C12、3T3-L1세포계，검측기24 h후적쌍형광소매활성。실험결과획득료관령우MyoD옥기인계동자，기서렬장도위993 bp，병성공구건료MyoD옥계동자보고질립；쌍형광소매활성검측표명pGL3-MyoD옥재소서C2C12세포중적표체시pGL3공재체40.65배，재소서3T3-L1세포중적표체시공재체적1.13배，MyoD옥계동자재소서C2C12세포중적표체고우3T3-L1세포(**p<0.01)。결과표명관령우MyoD옥기인계동자구유계동활성，재소서골격기세포중특이성표체。
To verify the promoter activity of the MyoD I gene by cloning promoter from the Guanling cattle, ac-cording to the MyoD I gene sequence of cattle in GenBank, the PCR primers were designed. The gene promoter was amplified by using PCR technology to construct the recombinant cloning vector pUCmT-MyoD I , and then identi-fied the positive clones by the PCR amplifying, restriction enzymes analysis and bioinformatics analysis. The report plasmid pGL3-MyoD I was constructed and transfected into the C2C12 and 3T3-L1 cells of mouse to detect the du-al luciferase activity after 24 hours. We obtained the promoter of MyoD I was obtained, and its sequence length was 993 bp. The report plasmid of MyoD I gene promoter was constructed successfully. The expression of pGL3-MyoD I was 40.65 times of pGL3 empty vector in mouse C2C12 cell, and was 1.13 times in 3T3-L1 cell through the method of relative activity of luciferase, which indicated that the expression of MyoD I gene promoter in C2C12 cell was higher than that of 3T3-L1 cell in mouse (**p<0.01). The results showed that the MyoD I gene promoter of Guanling cattle contains the promoter activity and can specifically express in skeletal muscle cells.
