CLONING AND CHARACTERIZATION OF H + -PYROPHOSPHATASE AND Na + /H + ANTIPORTER GENES FROM LEPTOCHLOA FUSCA AND THEIR EXPRESSION IN TOBACCO

Abstract

Leptochloa fusca L. is a halophyte plant from the Poaceae family and is also locally known as kallar grass. It is a perennial, summer-growing, forage grass that is now cultivated in many parts of the Pakistan and India. It is highly tolerant to salinity, sodicity, water logging and high soil pH. Plants have evolved a variety of adaptation mechanisms against salt stress, such as; restricting the uptake of environmental Na + ; increasing the efflux of Na + from the cell; and sequestering Na + into the large intracellular vacuole to reduce Na + accumulation in the cytosol. Compartmentalization of the Na + into vacuoles can be accomplished by the action of Na + /H + antiporters in the vacuolar membrane. They catalyze the exchange of Na + for H + across tonoplast membranes using the proton gradient generated by the vacuolar H + -ATPase and H + -pyrophosphatase (PPase). The function of the NHX1 antiporter depends upon free H + provided by protons pumps. The objectives of the study were to isolate H + -pyrophosphatase and Na + /H + antiporter genes from Leptochloa fusca and characterize for salt and drought tolerance. During present study the full length H + - PPase and NHX1 genes were isolated from cDNA of L. fusca using degenerate primers generated from corresponding sequences of plants of the Poaceae family. The L. fusca H + -PPase (LfVP1) cDNA contained an uninterrupted open reading frame of 2,292 bp, coding for a polypeptide of 764 amino acids. The LfVP1 sequence showed 91% identity with Z. mays H + -PPase, 90 % with S. bicolor and 80 % with A. thaliana H + -PPase. The L. fusca NHX1 cDNA contained an uninterrupted open reading frame of 1,623 bp coding for a polypeptide of 541 amino acids. The L. fusca NHX1 gene sequence showed 88 % identity with S. bicolor and 87 % with Z. mays genes. The LfVP1 and LfNHX1 genes were characterized using various online bioinformatics tools; hydrophobicity plots; 2 dimensional transmembrane structures; and protein 3 dimensional structures. The LfVP1 and LfNHX1 genes were cloned under the control of Gal promoter in a Gateway ® yeast expression vector and transformed in rg9 (control); ena1 and ena1;nhx1 yeast mutants. Yeast complementation assay on hygromycin plates showed that overexpression of the LfVP1 and LfNHX1 genes suppressed the hygromycin susceptibility phenotype in yeast mutants. The LfVP1 and LfNHX1 were also cloned under control of the 35S, 2X-35S, ZmUbi and OsAct promoters using the Gateway ® technology. The LfVP1 and LfNHX1 genes were transferred in tobacco through Agrobacterium mediated plant transformation under xvicontrol of the 35S promoter and characterized for salt and drought tolerance. The LfVP1 and LfNHX1 transgenic lines showed higher levels of relative water contents, stomatal conductance, net photosynthetic rate, membrane stability index and more negative value of leaf osmotic potential as compared to wild type control plants. The LfVP1 and LfNHX1 transgenic plants were able to germinate and maintain their growth at to 200 mM and 250 mM NaCl. The LfVP1 and LfNHX1 transgenic plants also showed better germination at 2 mg L - Basta ® ( glufosinate-ammonium ) . The LfVP1 and LfNHX1 genes were also transformed in wheat under ZmUbi promoter. Putative transgenic plants were confirmed through PCR amplification and leaf bioassays.