Expression of a vascular na+
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The document reports on the isolation of a vacuolar Na+/H+ antiporter gene from alfalfa, called MsNHX1. The gene was found to encode a functional Na+/H+ antiporter when expressed in yeast. Arabidopsis plants were genetically engineered to overexpress MsNHX1, which resulted in enhanced salt tolerance of the transgenic plants compared to wild-type plants. The study demonstrates that overexpressing the alfalfa MsNHX1 gene improves salt tolerance in Arabidopsis.
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- 1. EXPRESSION OF A VASCULAR NA+/H+ANTIPORTER GENE OF ALFALFA ENHANCES SALINITY TOLERANCE IN TRANSGENIC ARABIDOPSIS By: Nandini Parkavi.S 15BBT028 III-year BIOTECH Reg no: 201507035 AN Bao-Yan1,2,**, LUO Yan1,**, LI Jia-Rui2 , QIAO Wei-Hua3 , ZHANG Xian-Sheng1,*, and GAO Xin-Qi1,
- 2. ALFALFA..... Alfalfa, Medicago sativa also called lucerne, is a perennial flowering plant---pea family (Fabaceae) cultivated as an important forage crop in many countries around the world
- 3. ARABIDOPSIS....... Arabidopsis (rockcress) is a genus in the family Brassicaceae. They are small flowering plants related to cabbage and mustard.
- 4. INTRODUCTION.... The growth and development of plants are affected by drought, salinity, cold, and other adverse environmental factors, which cause great losses in yield. Salt can cause ion toxicity and osmotic stress and subsequently physiological drought
- 5. SO... Salt-tolerant plants transport Na+ into vacuoles or remove Na+ out of cells from the cytoplasm by which plants avoid the toxic effects of Na+ in the cytosol and maintain osmotic balance.
- 6. IN THIS PAPER.... They isolated a Na+/H+ antiporter gene, designated MsNHX1, from alfalfa. In transient transfection assay, the MsNHX1- GFP fusion protein was targeted to the vacuolar membrane. Transfection is the process of introducing naked or purified nucleic acids into eukaryotic cells.
- 7. PLANT MATERIALS..... The alfalfa seeds and The sterilized Arabidopsis seeds were planted
- 8. ISOLATION OF NA+/H+ ANTIPORTER GENE FROM ALFALFA..... Total RNA isolation. On the basis of the conserved region of Na+/H+ antiporter gene, primers were designed . 5'-CA(T/C)TA(C/T)AC(C/T)TGGCA(C/T)AA(C/T)GT-3' 5'-CAT(G/C)AG(A/C/)CC(A/G/T)GCCCACC(A/G/T)AT-3'. The resulting cDNA clone, 309 bp in length, has high homology to A. thaliana. 5' RACE for Rapid Amplification of cDNA Ends. 3' MsNHX1 cDNA segment was prepared using a 3' RACE technique
- 9. CONSTRUCTION OF PBI121-MSNHX1-GFP AND LOCALIZATION ANALYSIS OF MSNHX1 USING GFP... MsNHX1 cDNA was amplified usins primers. The region was inserted into the BamH1 site of pBI121-GFP vector. The fusion product was transformed into onion (Allium cepa L.) by Agrobacterium-mediated transformation.
- 10. SOUTHERN BLOT ANALYSIS.... Ten micrograms of genomic DNA were digested using EcoR I and EcoR V restriction enzyme at 37°C overnight. And the southern blot analysis was done.
- 11. NORTHERN BLOT ANALYSIS.... Three-week-old alfalfa seedlings were treated with 200 mmol L−1 NaCl. Young leaves and roots were harvested after 0, 3, 12, 24, and 48 h and were frozen in liquid nitrogen and stored at –70°C. Total RNA was extracted using total RNA isolation system as mentioned above. Northern blotting was carried out . The MsNHX1 probe was synthesized using the same template as the Southern blotting analysis.
- 12. FUNCTIONAL EXPRESSION OF MSNHX1 IN YEAST..... The complete MSNHX1 coding region was subcloned into the yeast expression vector pYES2.
- 13. PLASMID CONSTRUCTION AND AGROBACTERIUM- MEDIATED TRANSFORMATION...... The MsNHX1 gene in pGEM-T Easy Vector was digested using Xba I and Sal I and subcloned into pBI121 vector. Arabidopsis transformation was conducted using floral dip method.
- 14. MOLECULAR CHARACTERIZATION OF TRANSGENIC PLANTS.... Total RNA was isolated from leaves of T1 transgenic plants and wild type plants.
- 15. SALT TOLERANCE TEST OF TRANSGENIC ARABIDOPSIS PLANTS..... The wild-type plants were grown on GM medium containing 0, 100, and 200 mmol L−1 NaCl.
- 16. Isolation of MsNHX1 gene from alfalfa. MsNHX1 gene organization in alfalfa genome and its expression pattern under salt stress condition was studied. MsNHX1 is a functional Na+/H+ antiporter in yeast cells.
- 17. Alignment of amino acid sequences of NHX1 from several plant species
- 18. PHYLOGENETIC TREE OF PUTATIVE NA+ /H+ ANTIPORTER PROTEINS. MSNHX1 (AY513732, BOXED), MEDICAGO SATIVA;
- 19. VACUOLAR MEMBRANE LOCALIZATION OF PBI121-MSNHX1-GFP TRANSIENT EXPRESSION IN ONION EPIDERMAL CELL..
- 20. EXPRESSION OF MSNHX1 IN YEAST NHX1 MUTANT AFTER COMPLETE MSNHX1 WAS SUBCLONED INTO PYES2, THE PYES2 EMPTY VECTOR AND PYES2-MSNHX1 CONSTRUCT WERE TRANSFORMED INTO ATX3 (ǺNHX1)
- 21. MOLECULAR ANALYSIS OF MSNHX1 GENE....
- 22. SALT TOLERANCE ANALYSIS OF WILD-TYPE AND TRANSGENIC ARABIDOPSIS PLANTS OVEREXPRESSING MSNHX1......
- 23. PHYSIOLOGICAL ANALYSIS OF TL1 AND WILD-TYPE (WT) PLANTS UNDER TREATMENT WITH 200 MMOL L−1 NACL....
- 24. CONCLUSIONS.... Thus from alfalfa, a vacuolar Na+ /H+ antiporter gene, MsNHX1 was isolated, whose functions in Na+ transport. The transcription of MsNHX1 was induced under salt stress, and the overexpression of MsNHX1 in Arabidopsis improved the salt tolerance of the transgenic plants.