生物化学与生物物理进展
生物化學與生物物理進展
생물화학여생물물리진전
PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS
2009年
5期
556-565
,共10页
农杆菌%反向可选择性标记基因%同源重组机理%无标记突变体
農桿菌%反嚮可選擇性標記基因%同源重組機理%無標記突變體
농간균%반향가선택성표기기인%동원중조궤리%무표기돌변체
Agrobacterium tumefaciens%counterselectable marker gene%homologous recombination mechanism%unmarked mutant
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.
農桿菌已經用作許多生物過程研究的模型細菌,為瞭解析這些生物過程的分子機理,對農桿菌的某些基因進行突變就顯得非常重要.以自殺性基因sacB作為反嚮可選擇性標記基因,利用同源重組的原理,建立瞭一種可對農桿菌基因進行準確插入、刪除和位點置換的突變方法,所穫突變體不帶任何不需要的外源DNA序列.通過詳細研究同源序列的長度對農桿菌同源重組效率和突變體產生概率的影響,以及對農桿菌中的同源重組機理的分析,提齣瞭優化該突變體產生方法的方案,即通過設計不等長的上下遊同源序列和選擇其中一種類型的單交換重組體來篩選二次交換重組體的方法,可以顯著地提高理想突變體的產生概率.研究結果對如何提高突變體的產生概率和減少突變體篩選的工作量具重要的參攷價值.利用該方法成功地穫得瞭兩箇基因被同時刪除而且不含抗性標記的農桿菌突變株.
농간균이경용작허다생물과정연구적모형세균,위료해석저사생물과정적분자궤리,대농간균적모사기인진행돌변취현득비상중요.이자살성기인sacB작위반향가선택성표기기인,이용동원중조적원리,건립료일충가대농간균기인진행준학삽입、산제화위점치환적돌변방법,소획돌변체불대임하불수요적외원DNA서렬.통과상세연구동원서렬적장도대농간균동원중조효솔화돌변체산생개솔적영향,이급대농간균중적동원중조궤리적분석,제출료우화해돌변체산생방법적방안,즉통과설계불등장적상하유동원서렬화선택기중일충류형적단교환중조체래사선이차교환중조체적방법,가이현저지제고이상돌변체적산생개솔.연구결과대여하제고돌변체적산생개솔화감소돌변체사선적공작량구중요적삼고개치.이용해방법성공지획득료량개기인피동시산제이차불함항성표기적농간균돌변주.
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.