产生无标记农杆菌突变体方法的建立及优化

农杆菌已经用作许多生物过程研究的模型细菌，为了解析这些生物过程的分子机理，对农杆菌的某些基因进行突变就显得非常重要．以自杀性基因sacB作为反向可选择性标记基因，利用同源重组的原理，建立了一种可对农杆菌基因进行准确插入、删除和位点置换的突变方法，所获突变体不带任何不需要的外源DNA序列．通过详细研究同源序列的长度对农杆菌同源重组效率和突变体产生概率的影响，以及对农杆菌中的同源重组机理的分析，提出了优化该突变体产生方法的方案，即通过设计不等长的上下游同源序列和选择其中一种类型的单交换重组体来筛选二次交换重组体的方法，可以显著地提高理想突变体的产生概率．研究结果对如何提高突变体的产生概率和减少突变体筛选的工作量具重要的参考价值．利用该方法成功地获得了两个基因被同时删除而且不含抗性标记的农杆菌突变株．

Development and Optimization of Method for Generating Unmarked A. tumefaciens Mutants

Agrobacterium tumefaciens possesses many advantages as a model bacterium for the study of a wide variety of biological processes. Gene disruption or inactivation is a powerful and direct tool for investigation of in vivo gene functions. The intensive study ofA. tumefaciens has increased the need for simple and highly efficient procedures to manipulate its genome. The sacB gene was used as a counterselectable marker to develop a gene replacement procedure that allows precise insertion, deletion, and allele substitution of any gene sequence in A. tumefaciens without altering the genome in any other way. A kanamycin resistance (KmR) cassette was constructed to the suicide vector as the positive selection marker. The suicide plasmid containing DNA fragments homologous to the flanking sequences of the target gene was integrated into the recipient cell genome at the target gene locus by intermolecular homologous recombination, generating the KmR-single cross-over colonies. The effect of homologous sequence length on the intermolecular homologous recombination was analyzed. The second cross-over colonies generated by intramolecular homologous recombination occurring between two tandem repeats were simply screened out by counter-selection of sacB. Data showed that the intervening sequence length between two repeats significantly affected the intramolecular homologous recombination frequency in A. tumefaciens, indicating that A. tumefaciens adopted the homologous recombination mechanism similar to that in E. coli. All these results demonstrated that investigators could minimize the numbers of colonies to be analyzed and reduce the overall workload by optimizing the relative length of two homologous fragments and using the specific type of single cross-over transformants for screening the second cross-over event. This mutagenesis strategy had successfully been used to generate the double unmarked △vbp2△vbp3 mutant in two A. tumefaciens strains.