Characterization and Over-Expression of Scdr1 Endogenous Gene to Enhance Abiotic Stress Tolerance in Sugarcane

Changing climatic conditions in combination with increasing salinity and drought stress is a real challenge in developing robust crop plants with sustainable growth and production. Pakistan is the fifth top-most grower but 11th largest producer of sugarcane. Abiotic and biotic stresses are major impediments lowering its yield. Advancements in molecular approaches provide a swift solution to alleviate these stresses. Sugarcane has complex genome and various endogenous stress responsive genes have potential to combat abiotic stresses. A novel stress responsive gene Scdr1 was charcaterized in indigenous elite sugarcane genotypes. Phylogenetic analysis revealed prevelance of thisstress responsive gene in various monocots including maize, sorghum, wheat and rice etc. Seven indigenous sugarcane genotypes (S2003-US-127, S2006-US-272, CP-77400, CPF-246, CPF-247, S2003-US-778 and CPF-248) were grown in pots and exposed to salt stress (170 mM). Expression analysis of Scdr1 gene was carried out in the aforementioned genotypes by realtime quantitative PCR. It appeard to be salt stress inducible as expression of the gene was higher in salt treated plants as compared to plants growing under normal conditions. However, its expression was maximum in CP-77400 and minmum in genotypes CPF-246 and S2003-US-778 under salt stress. For the functional characterization and localization of the Scdr1 protein, it was translationally fused with GFP as well as co-transformed with endoplasmic reticulum fluoresent marker (CD3-959-ER-rk). Moving protein bodies were observed in the cytoplasm of N. benthamiana leaf cells under confocal laser scanning microscope but appropriate location of the protein was not traceable, perhaps owing to overexpression of the protein. Y2H cDNA library was constructed to identify the potential interactors of this stress responsive protein. It appeared to interact with the proteins involved in abiotic as well as biotic stress tolerance. To validate, its role in biotic stress tolerance, red rot resistant (CPF-246, S2006-US-658) and susceptible genotypes (S2003-US-127, SPF-234) were inoculated with Colletotrichum falcatum and quantitative expression analysis was performed. Scdr1 was significantly up-regulated in resistant genotypes whereas down-regulated in susceptible genotypes. Hence, in addition to abiotic stress tolerance, Scdr1 was proposed to play some crucial role in biotic stress tolerance as well. Considering the importance of Scdr1 gene in biotic and abiotic stress tolerance, it was synthetically developed and cloned under maize ubiquitin promoter (Ubi), nos terminator and was physically linked with selectable marker gene (bar) cloned under CaMV35 S promoter and terminator. Scdr1 construct (Ubi P:Scdr1:nos T::35S P:bar:35ST) was transformed into tobacco as well as in dark proliferated sugarcane calli. Bombarded calli/ leaf discs were shifted to regeneration medium supplemented with PPT. Putative transgenic plants were confirmed with PCR using maize ubiquitin promoter and nos terminator specific primers. Quantitative real time PCR was performed to seek for transcript level of the recombinant Scdr1. It appeared to be 1.4 folds higher in the transformed plants as compared with un-transformed plants of the genotype S2003-US-778.Hence, sugarcane plants with over-expression of recombinant Scdr1 gene were developed which are expected to be better tolerant to abiotic and biotic stresses.