Pseudomonas Pseudomonas Xm Salt Tolerance Gene Cloning And Transformation

A new species (or strain) of balopMous bacterium has been found from thesaturation brine pooi in XIAMEN city, CHINA, and designated temporarily as Pseudomonas X~vt For cloning its halo-tolerant-related genes, the total genomic DNA of this bacternrn has been extracted and then digested by the restnction endonuclease Sau3A I After agarose gel electrophoresis, allthe DNA fragments ranged from l'-9kb were retrieved, purified and linked with the plasmid pUC19 digested by restriction endonuclease BamH I, so a partial DNA librazy of Pseudomonas XM has been established by using E.coli JMIO9 as receptors. Some of the transformants of the partial DNA libraiy were transferred to a selective LB medium containing Amp(lOO i.i g/ml) and NaC1(6%).Finally a few transformants which could grow in the medium mentioned above passed through this screening procedure. Fouthermore, we extracted one of the reconstruction plasmids from the transformants, and found that a DNA fragment about 8. 8kb in size was inserted into the plasinid. By subcloning this fragment, a 4. 2kb DNA fragment related to salt tolerance has been luckly obtained.So far we have recombined the 4.2kb DNA fragment with the gene integration platform system plasmid pUCR and transferred the recombinant plasniid pZYR into Synechocciccus sp. PCC 7942. Passing through the screening precedure, we have obtained transformant Synechococcus sp. PCC 7942 that raising its level of salinity tolerance ability. We found that the salinity tolerance ability is stabilizative through consecutive cultivating By the aid of two-dimensional gel eletrophorsis analysis, two special proteins have been found in the transformant Synechococcus sp. PCC 7942-50-26KD respectivlyIt is shown from the sequencing and analyses of the 4.2kb DNA fragment that three open reading frames(ORFs) may be involved in it The three ORFs are 1.4kb, 0.7kb and 0.5kb in size. According to the putative amino acid sequence of the three ORFs, they probably are the genes of NADH dehydrogenase II, phosphoglycolate phosphatase and transcriptional regulator.By the results of the experiments of transferring the 4.2kb DNA fragment into Synechococcus sp. PCC 7942, it can be speculated that there may possess an intact transcription structure. So, it had been introduced into the plasmid of pCAMBIA 1301 and fomied recombinant plasmid of pZYT. Soon afer we transfen~d the pZYT into the callus of Ipoaoea aqua tica, the result of GUS transient expression ~s apparently positive and Southern blotting hybridization analysis of transfomiant indicated that the foreign gene had been integrated into the genome of Ipoaoea aqua tica So we may say that the vegetable transgenic transfom'iant system has been established in our laboratory.