【摘要】 以低拷贝质粒pACYC184为载体,将hok/sok基因插入到载体BamHⅠ位点,构建重组质粒pACYC184-hok/sok稳定系统,同时构建hok/sok基因缺失突变重组质粒pACYC184-hok/sok(M)。通过对构建的重组菌进行连续传代和对不同代次的重组质粒进行SphⅠ酶切,分析hok/sok基因的引入对宿主细胞生长的影响。结果表明,重组质粒pACYC184-hok/sok在无抗生素筛选压力下连续传代,传至第135代时质粒稳定率仍是100%,且传代前后的质粒SphⅠ酶切图谱没有发生变化,DNA序列测定表明,插入的hok/sok基因没有发生任何突变。突变重组质粒pACYC184-hok/sok(M)传代前后的质粒SphⅠ酶切结果虽没有发生变化,但其相应的重组菌传至第15代,质粒几乎完全丢失。生长曲线的测定结果表明,与含pACYC184重组菌生长曲线相似,pACYC184-hok/sok重组菌生长较快,而pACYC184-hok/sok(M)重组菌生长缓慢。以上结果表明,含hok/sok稳定系统,其稳定性明显高于无hok/sok稳定系统的质粒pACYC184以及突变质粒pACYC184-hok/sok(M)。重组质粒pACYC184-hok/sok具有良好的结构稳定性和分离稳定性。更多还原

【Abstract】 The hok/sok gene was inserted into the low copy plasmid pACYC184 with its BamHⅠsite.The recombinant plasmid pACYC184-hok/sok was constructed,then transformed into E.coli DH5α.According to the recombinant plasmid DNA sequence of pACYC184-hok/sok,the special primers were designed for long PCR,in order to combine hok/sokΔ274T with the plasmid DNA sequence of pACYC184.Then the long PCR products self-ligated to develop another recombinant plasmid pACYC184-hok/sok(M),used as a control for pACYC184-hok/sok.To determine the segregational instabilities of the constructed plasmids,the two recombinant E.coli DH5αstrains with plasmids of pACYC184-hok/sok and pACYC184-hok/sok(M),and the control strain with pACYC184,were respectively cultured under the non-antibiotic condition,thus testifying the relations between plasmid loss and no antibiotics addiction during continuous division of host cells.The method was dedicated as follows:the recombinant bacterial strains were cultivated continuously in LB(cm+),and at regular intervals,appropriately diluted samples were spread on LB agar plates.The recombinant plasmids extracted from both the original strains and the 135th or 15th (as the recombinant strain containing pACYC184-hok/sok(M) was very unstable,only grown for 15 generations) generation strains were digested by SphΒ,additionally with the plasmid pACYC184 as a control.Considering whether the hok/sok PSK system effects host cell growth,we charted the growth curves of two recombinant strains as well as the genetic engineering bacteria containing pACYC184.The experimental results showed that,under the non-antibiotic condition,the recombinant DH5α(pACYC184-hok/sok) were stably maintained.135 generations;but the recombinant DH5o(pACYC184-hok/sok(M) ) was very unstable,only grown for 15 generations with 98%of plasmid loss.Hence,hok/sok PSK system can enhance the plasmid stability of pACYC184 obviously.Further restriction and sequencing analysis identified the consistency and correctness of pACYC184-hok/sok DNA sequence from both the original strains and the 135th generation strains.However,the similar results also happened to the recombinant plasmid pACYC184-hok/sok(M),it had little genetic importance because of the short generations maintaining by its host strain.Compared with the genetic engineering bacteria containing pACYC184,DH5α(pACYC184-hok/sok (M)) grew slowly during all stages of growth,which suggested that its low growth rate was closely correlated with plasmid instability.Whereas DH5α(pACYC184-hok/sok)grew more rapidly,its growth curve was similar to the control bacteria’s.Subsequently,we came to a conclusion that,the stability of hok/sok-carrying plasmid pACYC184-hok/ sok was obviously better than hok/sok-free plasmids of pACYC184 and pACYC184-hok/sok(M).The recombinant plasmid pACYC184-hok/sok has been evolved with good segregational stability.更多还原