铜绿假单胞菌整合子可变区长度对intⅠ1基因表达的影响

Impact of length of variable regions of integrons in Pseudomonas aeruginosa on expression of intⅠ1 gene

目的分析铜绿假单胞菌中Ⅰ类整合子可变区的长度对Ⅰ类整合酶基因(intⅠ1)表达水平的影响,以探讨整合酶表达的调控机制。方法将25株临床分离的Ⅰ类整合子阳性铜绿假单胞菌按可变区的长度分为A、B组(A组:1000～2000bp;B组:>2000bp),提取细菌的总RNA并逆转录为cDNA,用实时荧光定量PCR法测定两组细菌intⅠ1mRNA的相对表达量,并比较具有不同长度可变区的细菌中intⅠ1基因的平均表达水平。结果两组细菌中intⅠ1mRNA的相对表达量差异有统计学意义(P<0.05);A组细菌intⅠ1和16SrRNA的Ct值均值及⊿Ct值分别为:17.25、10.34、6.91,B组细菌intⅠ1和16SrRNA的Ct值均值及⊿Ct值分别为:18.91、9.47、9.45,⊿⊿Ct为5.81;A组细菌的intⅠ1mRNA平均表达水平是B组的5.81倍。结论在临床分离的Ⅰ类整合子阳性的铜绿假单胞菌中,短可变区组整合酶表达水平较高,而长可变区组整合酶表达水平较低,提示Ⅰ类整合子中整合酶的表达水平可能受整合子可变区基因盒数目的影响,从而影响整合子继续捕获基因盒的能力。

OBJECTIVE To study the impact of length of different variable regions of integrons in Pseudomonas aeruginosa on the expression of intⅠ1 gene so as to explore the mechanism of regulating the expression of integrase.METHODS Totally 25 strains of P.aeruginosa with intⅠ1 gene were divided into 2 groups according to the length of variable regions of their integrons（Group A： 1000-2000bp;Group B： above 2000bp）.The total RNA of these bacteria was extracted and cDNA was synthesized by reverse transcription PCR.The relative expression of intⅠ1 gene of different variable regions were detected by real-time fluorescent quantitative PCR.RESULTS P.aeruginosa strains in the two groups expressed intⅠ1 mRNA with different levels（P0.05）.For the group A,the average value of Ct intⅠ1 was 17.25,the average value of Ct 16S rRNA was 10.34,and ⊿Ct value was 6.91.For Group B,the average value of Ct intⅠ1 was 18.91,the average value of Ct 16S rRNA was 9.47,and ⊿Ct value was 9.45.For the two groups,⊿⊿Ct value was 5.81.The average level of intⅠ1 mRNA of bacteria in the group A was 5.81 folds as the group B.CONCLUSION Among the clinical isolates of P.aeruginosa with intⅠ1 gene,the bacteria in the group A have expressed more intⅠ1 mRNA than the bacteria in the group B did.It is suggested that the length of variable region（or the number of gene cassettes） may contribute to the expression of integrase,and thus contribute to the capacity of integrons for capturing gene cassettes.