pBI121-Lyz-GFP表达载体的构建及其对和田苜蓿愈伤组织的转化

利用分子克隆技术，将GFP基因片段插入到pBI121-Lyz多克隆区，构建了重组表达载体pBI121-Lyz-GFP。经SmaI与BamHI双酶切和PCR验证重组质粒，均能得到约750bp的目的片段，通过进一步测序证明：GFP基因已成功连接到pBI121-Lyz中，且重组质粒连接方向正确。利用冻融法将重组质粒导入农杆菌菌株LBA4404，用叶盘法转化和田苜蓿，筛选出具有卡那霉素抗性的愈伤组织，经PCR扩增和荧光显微镜检测证明重组基因已成功转化到和田苜蓿愈伤组织中。

Construction of pBI121-Lyz-GFP Expression Vector and Its Expression in HeTian Alfalfa

All experiment methods were performed according to the standard of molecular clone technology. The DNA fragment of GFP gene was inserted into the pBI121-Lyz to get the recombined eukaryotic expression vector pBI121-Lyz-GFP. Polymerase chain reaction (PCR) was used to proliferate GFP gene from pBI121-Lyz-GFP plasmid, producing an approximate 750bp band with restriction enzyme SmaI and BamHI at both sides of the DNA. The eukaryotic expression vector was also verified by nucleotide sequencing. Plasmid pBI121-Lyz-GFP was introduced into agrobacterium tumenfaciens strain LBA4404. Discs of HeTian alfalfa were transformed with LBA4404 strain. Kanamycin resistance was selected and induced to regenerate alfalfa callus. The observation of GFP expression in HeTian alfalfa callus with slides was carried out under the fluorescent microscope upon excitation of blue light. PCR amplification and the results of observation showed that the recombined plasmid was transformed into HeTian alfalfa callus successfully.