摘要
二亲本杂交实验通常要求受体菌不能携带有与质粒上相同的抗性.本次实验中受体菌铜绿假单胞菌(Pseudo-monas aeruginosa)和重组质粒pHN102都带有四环素(Tc)抗性.考虑到质粒pHN102导入受体菌后至少带有两个Tc抗性基因片段,转移接合子的抗性会增强,所以提高四环素浓度至20μg/mL,并利用pHN102上携带的luxAB发光酶基因标记受体菌筛选转移接合子,并设立对照菌株.通过抽提质粒、琼脂糖凝胶电泳检测,确定pHN102成功转入P.aeruginosa中.转移接合子经4次转接后,质粒仍可以稳定存在且能够稳定表达.P.aeruginosa(pHN102)的发光动力学曲线表明,加入底物20min后,发光强度趋于稳定.经液体培养稀释后进行发光度和活菌数测定,发现二者呈显著的线性关系(r=0.994).
Bi-parental mating requested the introduced plasmid and recipient strain could not contain the same resistance. In this experiment, the recipient strain Pseudomonas aeruginosa and the introduced plasmid pHN102 both contained tetracycline resistance. Introduction of pHN102 into P. aeruginosa and two fragments of tetracycline resistance would result in a higher resistance in transconjugant. Transconjugants were screened by increasing tetracycline to 20 μg/mL and identified as luminescence of transconjugant due to expression of luxAB in plasmid pHN102, as well as reference strain was set. The above method was successfully used to obtain transconjugant, and plasmid pHN102 could stably exist and express after the transferring of culture was done for four times. Plasmid extraction and agarose gel electrophoresis were applied to examine the transfer of pHN102 into P. aeruginosa. The results of bioluminescence kinetic curves of P. aeruginosa （pHN102） indicated that luminous intensity of P. aeruginosa （pHN102） tended to be stable by the addition of substrates after 20 minutes. There was a significant linear relation between luminous intensity and viable cell number of P. aeruginosa （ pHN102 ） （ r = 0.994）. Fig 4, Tab 2, Ref 6.
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2007年第1期93-96,共4页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金资助项目(No.20377018)~~
关键词
铜绿假单胞菌
luxAB
二亲本杂交
发光
Pseudomonas aeruginosa
luxAB gene
hi-parental mating
luminescence
