本研究利用Red/ET DNA重组技术对实验室已有的含鼠李糖基转移酶基因RhlAB的分泌表达载体pBBR1gentaRhlAB进行修饰，将3种不同强度组成型启动子（Papra、Ptac和PrhaB）成功替换了RhlAB基因在铜绿假单胞菌中的原始启动子，成功获得了表达载体pBBR1gentaPapraRhlAB，pBBR1kanPtacRhlAB和pBBR1kanPrhaBRhlAB，并将这些表达载体分别在防御假单胞菌Pf5中异源表达，通过LB培养基发酵42 h后，Pf5/pBBR1gentaRhlAB的鼠李糖脂产量为17.56 mg/L，而Pf5/pBBR1gentaPapraRhlAB，Pf5/pBBR1kanPtacRhlAB和Pf5/pBBR1kanPrhaBRhlAB分别是11.135 mg/L，441.135 mg/L，557.764 mg/L，启动子优化后产量分别是原始启动子的0.63，25.12和31.76倍。对发酵产物进行高效液相色谱质谱联用技术分析，共检出相对含量变化的4类质核比不同的鼠李糖脂同系物。并通过实时荧光定量PCR检测RhlAB基因的表达量，发现启动子替换为Ptac和PrhaB后RhlAB基因表达量分别是原始启动子的2.16和2.77倍。本研究初步实现了RhlAB基因在Pf5中的表达，发现组成型启动子Ptac和PrhaB比RhlAB的原始启动子表达效率更高，可为异源合成鼠李糖脂提供重要参考。

In this research, the recombinant expression vector pBBR1gentaRhlAB containing the rhamnosyltransferase gene (RhlAB)was modified via Red/ET homologous recombineering. Then, three recombinant expression vectors pBBR1gentaPapraRhlAB，pBBR1kanPtacRhlAB and pBBR1kanPrhaBRhlAB with three different constitutive promoters (Papra、Ptac and PrhaB)were constructed by subcloning technology，and then we electrotransformated the resultant expression vectors into P.protegens Pf5．When the flask in LB medium was fermented to 42 h, the rhamnolipid concentration of Pf5/pBBR1gentaRhlAB supernatant was 17.56 mg/L, with that of Pf5/pBBR1gentaPapraRhlAB, Pf5/pBBR1kanPtacRhlAB and Pf5/pBBR1kanPrhaBRhlAB supernatant being 11.135 mg/L, 441.135 mg/L and 557.764 mg/L, resulting in 0.63，25.12 and 31.76 fold variation after promoter optimization. High performance liquid chromatographymass spectrometry (LCMS/MS)analysis showed that there were total 4 rhamnolipid derivatives in the Pf5 engineering bacteria. Furthermore, the RhlAB gene expression was later examined by qRTPCR with the result showed that the expression level of RhlAB gene after Ptac and PrhaB promoter was 2.16 and 2.77 fold higher than that of the original one. Generally, this study preliminarily realized the expression of RhlAB gene in Pf5, and found that the constitutive promoter Ptac and PrhaB was more efficient than the original promoter which could provide a significant reference  with significance for heterologous biosynthesis of rhamnolipid.