摘要
采用随机克隆、功能筛选、逐次排除和同源比较的基因克隆新策略进行克隆假单胞菌(Pseudomonassp.cn4902)磷酸甘油磷酸酯酶基因的研究。结果表明,该结构基因长819bp,与铜绿假单胞菌的磷酸甘油磷酸酯酶基因的核苷酸一致性达61.5%,氨基酸同源性为56.2%。该基因已输入GenBank数据库,收录号AF348165。将该基因转化大肠杆菌,受体菌在含NaCl1.0mol/L的培养基中甘油含量升高2.9倍,最终菌浓度提高3.6倍。可见这是一个与生物耐盐性相关的主基因,以其转化、培育耐盐农作物的前景十分光明。
It is well known that, phosphoglycolate phosphatase (PGPase) is a key enzyme for converting glycerol phosphate into glycerol, responsible to salt tolerance in some organisms, especially in algae (Ginzburg, 1987). Nowadays, gene-encoding of PGPase has been performed for halophilic strain Pseudomonas sp. cn4902 isolated from a salt pool of extremely high concentration of NaCl, in Xiamen City, Fujian, by a functional gene screening technique described previously (Liu et al, 2004). Based on the knowledge that the PGPase is critical for the ability of salt tolerance in some halophilic organisms, our cloning strategy was optimized as follows. Total genomic DNA of Pseudomonas sp. cn4902 was extracted gently and digested partially by a restriction enzyme, e.g. Eco RI, and then run an agarose gel electrophoresis. Then the DNA fragments ranged from 1? ?kb were retrieved and linked with the linear plasmid pUC19 digested by the same enzyme. The reconstructed plasmids were found and transformed to E.coli JM101. All the transformants were inoculated on the LB agar media containing 6.0% NaCl to find the salt tolerance colonies. The survived colonies were selected and cultured in liquid salt media again to extract the reconstructed plasmids that consist of gene(s) responsible for salt tolerance. After digesting and subcloning step by step, a short DNA fragment (open reading frame) was finally found. Sequencing of the insert revealed an 819bp DNA with an open reading frame encoding a peptide of 272 amino acid residues. Both the DNA and its putative protein sequences were used with the “Search the Conserved Domain Database” using the Blast search of the GenBank by a bioinformatic approach, we found that they are highly homologous to the gene of phosphoglycolate phosphatase of Pseudomonas aeruginosa (61.5% and 56.2% at DNA and amino acid levels, respectively). The results mentioned above indicate that this short sequence may code for a phosphoglycolate phosphatase (PGPase) in Pseudomonas sp. cn4902. To satisfy this guess, we linked the gene with a high expression temperature control plasmid pBV220 and transferred them into E.coli ER2566. New transformants were found and cultivated in a liquid salt medium until OD_ 600 to 0.9—1.0 and then all the proteins and glycerol content of the transformants were detected. A clear protein band of about 28kD, which matches to the product of the cloned gene, could be seen after SDS-PAGE electrophoresis. On the other hand, it was showed that glycerol content in the transformants was 2.9 times more than control. Furthermore, the characteristic of salt tolerance of the transformant can be observed better in its liquid culture. The growth vs time plot was built by inoculating both transformant and control in liquid LB media supplied with 1.0mol/L NaCl and shaking at 37℃, monitored by checking OD every 6h in 48h growth period. The growth curve was repeated three times. We found that the transformant was clearly out performed than control by the phenomena that the former grew 6h after inoculation, while the latter did so much slower after 24h. Finally, the growth rate and its final bacteria concentration were much greater than the control group, which is 3.6-folds higher. This result indicated the function of the transgene by raising salt tolerance level of the transformants. It is believed that the gene we cloned is the PGPase gene of Pseudomonas sp. cn4902. GenBank has included it with access number AF348165.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2005年第4期313-318,共6页
Oceanologia Et Limnologia Sinica
基金
国家科技部转基因植物专项基金资助
J00B014号
厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室项目资助
2004106号。
关键词
假单胞菌
磷酸甘油磷酸酯酶
耐盐性
甘油
基因克隆
Pseudomonas sp., Phosphoglycolate phosphatase, Salt tolerance, Glycerol, Gene clone
