Cloning And Functional Analysis Of A LbGRP Gene From Limonium Bicolor

Drought,high-salt and low temperature are adverse environment condition that affect the growth of plants and productivity of crops. It has been reported that LbGRP(glycine-rich RNA-binding protein) plays an important role in resisting drought, high-salt and low temperature. The gene (LbGRP) had been cloned from Limonium bicolor, a tree species with high tolerant ability to extreme environmental conditions including saline, drought and freezing. In this study, the LbGRP gene was heterogenously expressed in yeast to test its resistant abilities. Meanwhile, the LbGRP gene was transformed into tobacco.genome by Agrobacterium-mediated transformation method. Then, the physiological traits were determined to examine the functions of the exogenous LbGRP gene in tobacco under saline stress. The results showed that:(1) Containing a Full-length cDNA sequence of glycine-rich RNA-binding protein was cloned from a cDNA library of Limonium bicolor. We examined the expression pattern of the LbGRP gene in leaves and roots of L. bicolor in response to NaCl,KCl,NaHCO₃,Na₂CO₃,low temperature and PEG stresses at different time points by real time RT-PCR. The results showed that LbGRP could be induced by low temperature treatment in leaves and roots, But the expression of LbGRP was inhibited by NaCl treatment in both leaves and roots of L. bicolor.(2) The recombinant plasmid pYES2-LbGRP was constructed by inserting LbGRP gene to yeast expression vector pYES2. The recombinant vector was transformed into Saccharomyces cerevisiae (INVSc1 strain). PCR results indicated that the recombinant vector had been transformed into the yeast successfully. Furthermore, Northern bloting indicated that the exogenous gene can be transcript correctly.(3) The recombinant yeast INVScl(pYES2-LbGRP) was treated under NaCl, KCl, Na₂CO₃, NaHCO₃ ,PEG and low-tempertuate stress. The results revealed that the stress-resistance of recombinant yeast INVSc1 (pYES2-LbGRP) yeast was better than control strain. These indicated that the ability of stress-resistance of recombiant yeast had been enhanced due to over-expression of LbGRP gene.(4) LbGRP gene had been integrated into tobacco. tobacco via genome by Agrobacterium-mediated transformation systems. Twenty independent kanamycin -resistant shoots were obtained , detection nine of them and nine shoots showed positive result in PCR assay. Then Northern blotting indicated that exogenous gene can be transcript correctly in transgenic poplar plants.(5) Contents of malondialdehyde(MDA), activity of superoxide dismutase(SOD), activity of peroxidase(POD), Catalase(CAT), Proline and total soluble protein of the transgenic plants were examined and analysised under NaCl stress. The result shows: when the transgenic lines were treated with NaCl, the content of activity of superoxide dismutase(SOD), activity of peroxidase(POD), Catalase(CAT), Proline and total soluble protein of transgenic lines higher than nontransgenic lines, and the fourteenth transgenic line was best.All the results indicated that the expression of LbGRP gene can improve the resistant ability of plant salt and drought environment. Meanwhile the fourteenth transgenic plant was best in stress.