The Preliminary Salt-Tolerance Study Of Transforming A Betaine/Proline Transporter Gene Bet/Prot2of Mangrove Into Rice

Mangroves are halophytic woody plant communities which growing in the intersection of land an sea.They are recognized as the salt-tolerant plants and have high resistant ability of seawater. In recent years, mangroves are commonly used as salt tolerance materials. Proline is one of the largest water soluble amino acids and has a strong hydrophobic capacity.Its hydrophobic end can combine with protein and the hydrophilic end can combine with water molecules.This structure make the protein bound more water to prevent the cells from dehydration under osmotic stress. Betaine is another common non-toxic osmoregulative substances which can protect the biological macromolecules in high electrolyte concentration from variance through the interaction with the protein. On the other hand betaine is one of the osmotic balance materials to maintain cell turgor pressure. We grew the largest acreage grain crops in China, which also is a staple source of more than65%China population. However, the large area of land salinization cut larger number of rice production.In this study,we amplified Betaine/Proline transporter gene(Bet/ProT2) from Avicennia marina.Using bioinformatics method to analysis the cDNA sequences of Bet/ProT2and the protein structure of Bet/ProT2.Transformed the35S-Bet/ProT2-GFP fusion plasmids with GFP reporter gene into onion epidermal cells by Agrobacterium tumefaciens-mediated method. According to the green fluorescence of the GFP protein under excitation light to determine the subcellular localization of the Bet/Pro T2.And further used Agrobacterium-mediated to transform Bet/ProT2into Nipponbare Japonica callus. We attained transformed seedlings after election of resistance callus, acclimatization and soil culture. Finally, through PCR identification of transformed seedlings, histochemical determination of H2O2and150mmol/L NaCl salt stress to detect the salt tolerance of the Transgenic rice. This study was designed to improve the salt tolerance of rice by genetic engingeering. To lay the foundation for salt tolerance mechanism study of Bet/ProT2gene. The study showed:.The CDS length of Bet/ProT2is1344b.The Bet/ProT2protein has447amino acid residues;the relative molecular weight is48647.9KDa;the theoretical isoelectric point PI=9.4;the protein is a hydrophobic protein with positive charge;.Through the bioinformatics analysis found that amino acid sequence homology of Bet/ProT2with the three proline transporter AtProTl、AtProT2、AtProT3of Arabidopsis is73%、71%、71%respectively; amino acid sequence homology of Bet/ProT2with the two proline transporter LeProT1、LeProT2of tomato is65%、67%respectively; amino acid sequence homology of Bet/ProT2with betaine/proline transporter BvBet/ProT of suger beet is71%.We can speculate that Bet/ProT2has a certain relationship with betaine/praline transporter. Protein Bet/ProT2has11transmembrane region, This sequence without signal peptide and its subcellular localization prediction showed that the protein locate out of chloroplasts, mitochondria and endoplasmic reticulum region. Under the fluorescenece confocal microscopy, the green fluorescence of onion epidermal cells with35S-GFP expression vector located on the plasma membrane and intracellular.while the gree fluorescence of onion epiderma cells with35S-Bet/ProT2-GFP fusion expression vector located only on the plasma membrane. These results showed that Bet/ProT2protein is a transmembrane protein, which located on the cytoplasmic membrane.The bioinformatics analysis and subcelluar localization illustrate that the Bet/ProT2protein is a betaine and proline transporter which locating in the plasma membrane.The PCR results showed that Bet/ProT2gene was integrated into the genome of Nipponbare Japonica and expressed.we have obtained5lines T2transgenic plants. The analysis of T2generation of Bet/ProT2transgenic rice showed that there is no significant effect between transgenic rice and the wide-type under the normal conditions.But due to the over expression of the Bet/ProT2gene, the transgenic rice can effectively use the betaine and praline from outside to grow better than the wide-type.When betaine and proline were contained in the medium at the same time or exist separately, transgenic rice grew normally in the liquid medium containing150mmol/L NaCl, while wild-type rice was obvious wither. The transgenic rice did not grew better than wild-type rice in the medium without betaine and proline after the salt treatment.When betaine and praline were contained in the medium with150mmol/L NaCl,transgenic rice leaves have less content of H2O2than that in wide-type leaves.These results suggested that the Bet/ProT2gene of mangrove can significantly improve the salt tolerance of the transgenic rice.