Cloning And Functional Analysis Of A Late Embryogenesis Abundant Protein Gene SpLea5 From Sesuvium Portulacastrum

Sesuvium portulacastrum,belonging to Sesuvium L. Aizoaceae and perennial creeping succulent herb associated mangrove,is a growth in the beach sand and the both sides of tidal flat of river estuaries.As the the particularity of living environment,the Sesuvium portulacastrum,a typical of non-secreting halophyte salt plant,can normally grow watered by seawater and complete life cycle.Meanwhile, the Sesuvium portulacastrum well adapted to high salinity, drought and heavy metal.In order to reveal the molecular mechanism on salt-tolerance of Sesuvium portulacastrum,basing the Suppression Subtractive Hybridization(SSH),a differentially expressed cDNA library from roots of Sesuvium portulacastrum under seawater induction was constructed. When screening the ESTs of library, the dequence of SpSSH 860 is highly homologous with Late Embryogenesis Abundant Protein 5 of plant.For further study the structure and function of corresponding gene of EST SpSSH 860,a novel Late Embryogenesis Abundant Protein 5 is cloned from Sesuvium portulacastrum and its structure,physiological function and expression profie were studied.The key results were as follows:1. By the RACE (Rapid-Amplification of cDNA Ends) techniques,a novel Late Embryogenesis Abundant Protein 5 is cloned from Sesuvium portulacastrum, designated as SpLea5 (The GenBank Accession number, HQ427488).The sequence analysis showed that theSpLea5 cDNA has a total length of 685bp with an open reading frame of 294 bp, which is predicted to encode 97 amino acid residues.The genomic DAN fragment corresponding to SpLea5 comprise of two exons and one intron.2. Bioinformatics analysis indicated that theSpLea5 cotains a conserved domain of W (A/V) PDP (V/I) TGYYRPE as a class-5 motif of Lea5.Using phylogenetic analysis,the deduced amino acid sequence shares the identity of 54%、52%、47%、44% and 41% with that from Tamarix androssowii,Populus suaveolens,Citrus unshiu,Jatropha curcas,Glycine max and Ricinus communis, respectively. The phylogenetic tree showed that the SpLea5 shared the closet evolutionary relationship with Tamarix androssowii.3. The expression of SpLea5 gene was determined by RT-PCR analysis,showing that the SpLea5 was constitutively expressed,but the expression level of roots was significantly higher than in leaves and stems,and the abiotic stress such as Seawater, NaCl, ABA and PEG, could trigger a significant induction of SpFBA in S.portulacastrum roots.So we conclude that the SpLea5 may be involved in responses to abiotic stress.4. In order to further investigate molecular mechanism of SpLea5 on abiotic stress,the recombinant expression vector pET-30a-SpLea5 was construct and the recombinant protein was overexpressed in BL21 Escherichia coli.At the same time, the pCAMBIA1304-SpLea5 plant overexpressing vector was constructed and 41 transgenic tobacco plants were obtained by agrobacterium-mediated leaf disc transformation methed.Resistance analysis tests showed that the tolerance of transgenic plants to drought and salt-stress was significantly improved.Conclusion:The expression of SpLea5 gene was determined by RT-PCR analysis,showing that the SpLea5 was constitutively expressed,but the expression level of roots was significantly higher than in stems and leaves. The abiotic stress such as Seawater, NaCl, ABA and PEG, could trigger a significant induction of SpLea5 in S.portulacastrum roots.So we conclude that the SpLea5 may be play an important role in completing its life cycle of Sesuvium portulacastrum under long-term seawater. The SpLea5-overexpressing transgenic tobacco plants were given higher tolerance to drought and salt-stress than the controls.