CAJ | 학술논문

采用PCR 技术从大鳞副泥鳅（ Paramisgurnus dabryanus ）基因组中克隆了β-actin 基因近端启动子片段DPRK1，并将该基因启动子片段DPRK1定向亚克隆到不含启动子的绿色荧光表达载体pEGFP-N1中，构建了重组荧光表达载体pDPRK1-EGFP。将该载体分别转染真核细胞293T、 CEL及DF1，检测绿色荧光表达情况。重组载体经NdeⅠ酶切线性化后，显微注射到斑马鱼（ Danio rerio）的受精卵中。序列分析显示该片段的长度为1282 bp，含167 bp的5′侧翼序列近端启动子区和共1115 bp的第1个外显子以及第1个内含子区。5′侧翼序列近端启动子含有CAAT box， CArG motif和TATA box，分别位于转录起始位点（＋1）上游的-94、-64和-31处。在将重组载体pDPRK1-EGFP转染293 T、 CEL及DF1的实验中观察到3种细胞都有很强的绿色荧光，在显微注射到斑马鱼受精卵的实验中也观察到绿色荧光，该结果表明大鳞副泥鳅β-actin基因近端启动子片段DPRK1具有效的启动功能，为下一步创制转基因泥鳅新品系提供了技术支撑。
채용PCR 기술종대린부니추（ Paramisgurnus dabryanus ）기인조중극륭료β-actin 기인근단계동자편단DPRK1，병장해기인계동자편단DPRK1정향아극륭도불함계동자적록색형광표체재체pEGFP-N1중，구건료중조형광표체재체pDPRK1-EGFP。장해재체분별전염진핵세포293T、 CEL급DF1，검측록색형광표체정황。중조재체경NdeⅠ매절선성화후，현미주사도반마어（ Danio rerio）적수정란중。서렬분석현시해편단적장도위1282 bp，함167 bp적5′측익서렬근단계동자구화공1115 bp적제1개외현자이급제1개내함자구。5′측익서렬근단계동자함유CAAT box， CArG motif화TATA box，분별위우전록기시위점（＋1）상유적-94、-64화-31처。재장중조재체pDPRK1-EGFP전염293 T、 CEL급DF1적실험중관찰도3충세포도유흔강적록색형광，재현미주사도반마어수정란적실험중야관찰도록색형광，해결과표명대린부니추β-actin기인근단계동자편단DPRK1구유효적계동공능，위하일보창제전기인니추신품계제공료기술지탱。
By using PCR amplification techniques , β-actin gene proximal promoter fragment , called DPRK1, was cloned from Paramisgurnus dabryanus genome.The DPRK1 was inserted into pEGFP-N1, as a result, a fluorescent protein ex-pression vector pDPRK1-EGFP was constructed.Those vectors were transfected into 293T, celland DF1 to observe the green fluorescence expressions respectively .Meanwhile , the vector was line-typed by NdeⅠenzyme and microinjected in-to the fertilized eggs of Danio rerio.The complete sequence of the promoter fragment DPRK 1 was 1 282bp including 5′flanking proximal promoter (167 bp) and another fragment consisting of first exon and first intron (1 115 bp).The 5′flanking proximal promoter sequence contained CAAT box , GArG motif and TATA box, located at -94, -64 and -31 sites of upstream sequence respectively .Secondly , the recombined vector pDPRK 1-EGFP using experiment in 293 T, celland DF1 cells showed strong green fluorescence in these cells .And the fertilized eggs of D.rerio microinjected line-typed vector pDPRK1-EGFP also expressed the green fluorescence .This experiment results showed that β-actin gene proximal promoter DPRK1 of P.dabryanus had positive translation initiation activity and lay laid foundation for producing new varie-ties of transgenic loach .