甘菊DFLEGFP高效融合蛋白表达载体的构建.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 2 DFL EGFP 王翊 付建新 戴思兰 (, 100083) : 增强型绿色荧光蛋白( enhanced green fluorescent protein, EGFP) 是一种优化的突变型 GFP, DFL 是从甘菊中分 离 的 LFY基因的同源序列为了研究 FL基因的功能和表达模式,研究利用小片段克隆法将 linker序列插入到 EGFP基因 5!端启始密码子前面,在 pBI121载体的 CaMV35S启动子的 3!端后面插入一段多克隆位点,成功地构建了 pBI FLEGFP表达 载体通过设计特异引物, 利用 PCR 技术扩增得了到拟南芥 LFY基因的启动子序列, 用粘性末端 PCR技术将 pBI FLEGFP 表达载体中 CaMV35S启动子替换成 LFY基因启动子, 构建成了 pLFY FLEGFP表达载体用含有 pBI FLEGFP和 pLFY FLEGFP质粒的农杆菌侵染洋葱表皮细胞,在荧光显微镜下分别用蓝光激发, 均观测到了荧光这一结果表明, 融合蛋白 FL EGFP表达载体构建成功, 同时还证明了通过 PCR技术克隆到的 LFY启动子序列具有启动子功能 : 小片段克隆 粘性末端 PCR 表达载体构建 LF Y 启动子 FL EGFP融合蛋白 Construction ofHighExpression of Fusion Protein VectorsHarboring EnhanceGreen Fluorescence Protein Gene andDFL Gene inD endranthem a lavandulfi olium W ang Y i Fu Jianxin ai Silan (Colleg e of Landscap eA rch itecture, B e ij ing F orestry Un iv ersity, B eij ing 100083) Abstrac:t Enhanced green fluorescent protein( EGFP) is an optmi izedmutant ofGFP, andD FL is the homologous gene ofLFY i solated fromD endranthem a lavandu lfi o lium In order to study the function and expression patterns of FL, the linkerwere inserted into the front of the 5! end initiation codon by themethod of small fragments cloning in order to construct the fusion protein, and themultiple cloning sitewere inserted into the 3! end of CaMV35S promoter in pBI121 vector, thus the expression vector of pBI FLEGFP were successfully constructed. esign the specific prmi ers to get the LFY gene promoter sequence in A ra b idop sis by PCR amplification. The CaMV35S promoter in the pBI FLEGFP expression vector was replaced by LFY promoter through the sticky end of the PCR technolo gy then the pL