Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis
Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis
复制标题
DOI：
10.1093/pcp/pcf175
复制
发表时间：
2002-12-01
影响因子：
4.9
通讯作者：
Saeki, K
中科院分区：
文献类型：
--
作者：
Hattori, Y;Omori, H;Saeki, K
为了有效利用莲花微共生菌Mesorhizobium loti MAFF 303099的基因组序列信息，揭示其基因功能，我们利用IncP广宿主范围载体构建了一个有序且相互重叠的粘粒文库。该文库由480个克隆组成，覆盖约99.6%的基因组，平均插入片段大小和重叠分别为26.9和11.1 kbp。分枝杆菌基因组loti由一条染色体和两个质粒组成。445个克隆的染色体（7，036，071 bp）覆盖率为99.68%，有4个缺口，其中2个克隆在E.杆菌大质粒pMLa（351，911 bp）被23个克隆完全覆盖，而小质粒pMLb（208，315 bp）被12个带有两个缺口的克隆覆盖98.85%。我们还制备了辅助质粒，以便于使用文库克隆的衍生物构建缺失突变体。作为一个试点实验，以发现含有新的共生基因的区域，构建了13个缺失突变体，总共缺乏180.5 kbp的基因组。所有的突变体都形成了明显正常的根瘤，并支持共生固氮，然而，一个缺乏5.3 kbp染色体区域的突变体，4，551，930 - 4，557，222，不产生正常的胞外多糖，这是通过在含有Calcofluor的培养基上的荧光来判断的。实验结果支持了该方法检测基因功能的有效性。
For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions.