Identification & Molecular Cloning of Abiotic Stiless Tolerant Gene(S) and Stress Induced Biochemical Changes in Lentil (Lens Cull Naris)

Abstract

Plant productivity is adversely affected by nature's wrath in the form of variou biotic and abiotic stresses. They cause losses worth hundreds of million dollars each year due to reduction in crop productivity and crop failure In fact they threaten the sustainabi lity of agricultural industry. Lenti I (Lens culinaris) an important cool season food legume, encounters numerous biotic and abiotic stresses. Lentil contains asset of genes/proteins which helps this crop to overcome abiotic stresses and identification of stress inducible genes/proteins are an important area of research. In the present study, primers for two abiotic stress tolerant genes namely Inositol and Alcohol Dehydrogenase (ADH) were designed by Fast-PCR and validated by PrimerBLAST online tool. Stress treatment at four water potentials (-3.0,-5.0,-7.5 and -10.0 bar) was given, germination in -3.0 and -5.0 bar occurred and cotyledons collected were used for genomic DNA isolation by C-TAB method. Then, Inositol and ADH were identified through PCR which resulted in amp Iicon sizes of -1 OOObp and --2000bp respectively. The gel eluted Inositol and ADH were cloned in pGEM-T easy and pTZ57R/T vectors respectively. Cloning was confirmed by colony PCR and restriction digestion approaches. The confirmed clones were got sequenced. The sequence(s) obtained of Inositol and ADH having I 042bp & 1343bp were submitted to NCBI after BLAST analysis. These sequences were analyzed through bioinformatics softwares viz. BioEdit, ORF finder, Mega5. Nucleotide composition and rnultiple sequence alignrnents obtained identified and phylogenetic trees were constructed. These nucleotide sequences were then translated to protein sequences as to know their total amino acid corn position and evolutionary relationship of proteins with other species. Biochemical studies in PEG-6000 induced stress sho\>ved increased MDA and proline content whereas decreased carbohydrate and protein content as cornpared to water treated control. From these studies it can be concluded that the genes conferring drought tolerance and tolerance to other abiotic stresses can provide foundation for scientific irnprovernent of the crop ~s productivity under stress conditions and contribute to irnprovernent and stabilization of crop yields.