Improvement of salt tolerance in transgenic potato plants by glyceraldehyde-3 phosphate dehydrogenase gene transfer.

In the previous experiment, we isolated and characterized glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of the oyster mushroom, Pleurotus sajor-caju. Expression levels of the GPD gene in the mycelia of P sajor-caju was significantly increased by exposing the mycelia to abiotic stresses, such as salt, cold, heat, and drought. We also showed that GPD confers abiotic stress resistance when introduced into yeast cells. The survival rate of the transgenic yeast cell that harbored the GPD gene was significantly higher when the yeast cells were subjected to salt, cold, heat, and drought stresses, compared with the yeast that was transformed with the pYES2 vector alone. In order to investigate the functional role of the P. sajor-caju GPD gene in higher plant cells, the complete P. sajor-caju GPD cDNA was fused into the CaMV35S promoter and then introduced into potato plants. Putative potato transformants were screened by using PCR. Twenty-one transformants were further analyzed with RT-PCR to confirm the expression of P. sajor-caju GPD. A RT-PCR Southern blot analysis revealed that 12 transgenics induced the P. sajor-caju GPD gene expression. A bioassay of these transformants revealed that the P. sajor-caju GPD gene was enough to confer salt stress resistance in the potato plant cell system. Results showed that P. sajor-caju GPD, which was continuously expressed in transgenic potato plants under normal growing conditions, resulted in improved tolerance against salt loading.

Isolation and characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase.

A 1.2-kb full-length cDNA sequence of a glyceraldehyde-3-phosphate dehydrogenase (GPD) gene was isolated from the mushroom, Pleurotus sajor-caju. The full-length cDNA of the GPD gene consists of 1248 nucleotides, predicted to encode a 36-kDa polypeptide consisting of 335 amino acid residues. Sequence analysis revealed that the GPD gene has more than 72-78% amino acid sequence homology with those of other Basidiomycetes. Expression of the GPD gene increased when P. sajor-caju was treated with various abiotic stresses, such as salt, cold, heat, and drought. There was an eightfold induction by drought treatment. Salt and cold stress induced four- and twofold induction of GPD gene expression, respectively. There was also a fivefold induction by heat stress. The GPD gene exhibits different expression patterns under different stress conditions. It reached its maximum expression level within two hours under cold or heat treatment. The mRNA levels of this gene increased proportionally to increasing treatment time under salt or dry conditions. Because the expression of GPD was significantly increased, we tested whether GPD could confer abiotic stress resistance when it was introduced into yeast cells. For this, a transgenic yeast harboring P. sajor-caju GPD was generated under the control of a constitutively expressed GAL promoter. The results from biofunctional analyses with GPD yeast transformants showed that GPD yeast transformants had significantly higher resistance to cold, salt, heat, and drought stresses.

Expression Vector for Zymomonas mobilis.

This study describes the construction of several useful cloning vectors which can be conjugated from Escherichia coli into Zymomonas mobilis at high frequency, approaching 10 per donor or recipient. These vectors contain a broad-host-range replicon and mob site from RSF1010, a chloramphenicol acyltransferase gene under the control of an enteric consensus promoter, and a second mob site (originally derived from RP4). The addition of this second mob site appears to be responsible for a 2-order-of-magnitude increase in the efficiency of transfer into Z. mobilis. Such vectors may be useful for other gram-negative bacteria in which conjugation efficiencies are low. These vectors are stably maintained in Z. mobilis with no detectable loss of plasmid after 50 generations in the absence of selective pressure. One of these, pLOI193, contains the tetracycline gene from pBR322 and associated cloning sites for insertional inactivation. Another, pLOI204, contains a Z. mobilis promoter immediately upstream from a BamHI site which can be used for cloning. This promoter has been shown to efficiently drive the expression of beta-galactosidase in both Z. mobilis and E. coli. This promoter fragment from Z. mobilis has been sequenced, and the site for transcriptional initiation in E. coli and Z. mobilis has been identified.