Cloning And Functional Analysis Of Phospholipid Hydroperoxide Glutathione Peroxidase Genes From Sesuvium Portulacastrum

Sesuvium portulacastrum (Aizoaceae), a mangrove plant from seashore, is a halophyte species well adapted to salinity and drought. It can grow normally in sea water and fresh water after domestication, all of which determines it is an ideal material for salt-tolerant mechanism research.To investigate the physiological and molecular mechanism of salinity tolerance in Sesuvium portulacastrum, we successfully constructed a differentially expressed cDNA library from roots of Sesuvium portulacastrum under seawater induction by Suppression Subtractive hybridization (SSH). When screening the library, one EST (which)is highly homologous to plant PHGPx was found. On this basis, this study examined the activity of PHGPx related with salt stress and H2O2 stress, the cDNA full length of PHGPx was cloned in Sesuvium portulacastrum and a more complete molecular information was obtained, meanwhile the organ-specific, salt and H2O2 Stress-related gene expression were studied. Major results were shown as follows:1. Determination of PHGPx activity in Sesuvium portulacastrum leaves showed that the SpPHGPx activity is related with NaCl and H2O2 in dose. Under the treatment of Endogenous Peroxide, the activity of SpPHGPx increased with Continuously Stimulated by NaCl, while H2O2 does not work. Accordingly, we speculated that H2O2 is not essential in NaCl-mediated signal pathway.2. Based on SpSSH713 EST sequence information, a phospholipid hydroperoxide glutathione peroxidase gene named SpPHGPx was cloned(GenBank accession number:GU479913) by the method of RACE PCR. The SpPHGPx gene has a 821bp full length cDNA sequences with an ORF of 513bp, which encode 170 amino acid residues with a total predicted molecular 18.78 kDa, a theoretical pâ… 7.62. The deduced amino acid sequences of SpPHGPx showed high identities of 86%, 87%,89%and 85% to those of the phospholipid hydroperoxide glutathione peroxidase from ice plant (Mesembryanthemum crystallinum, AAB61592), spinach (Spinacia oleracea, CAA46649), castor (Ricinus communis, XP-002509790) and poplar (Populus trichocarpa, XP₀02299536). Biology informatics analysis indicated that SpPHGPx contains 3 sub-conservative domains which are specific in eukaryotic phospholipid hydroperoxide glutathione peroxidase, with no obvious signal peptide at N-terminal side is a stable non-transmembrane hydrophilic protein. 3. The expression of SpPHGPx gene was determined by RT-PCR analysis. SpPHGPx was constitutively expressed, but the expression level was significantly higher than in roots and stems. Abiotic stress such as sea water, NaCl and H2O2 could induce a significant increase in the expression of SpPHGPx.4. Recombinant expression vector pET-30a-SpPHGPx was constructed successfully and the recombinant protein was expressed.22 plants of Transgenic tobaccos were obtained by leaf discs transformation with expression vector pCAMBIA1304-SpPHGPx and identified by PCR technique.Conclusion:1. Salt stress can increase the activity of SpPHGPx, SpPHGPx expressed in Sesuvium portulacastrum played a key role in response to salt stress; endogenous hydrogen peroxide scavenging assay showed that the salt stress signal transduction in Sesuvium portulacastrum may be associated with H2O2 non-dependent antioxidant defense pathway.2. The SpPHGPx gene encoded phospholipid hydroperoxide glutathione peroxidase was constitutively expressed in Sesuvium portulacastrum, but the expression level in leaves was significantly higher than in roots and in stems; sea water, NaCl and H2O2 could induce a significant increase in the expression of SpPHGPx.