Functional analysis and Characterization of Wound Responsive Genes from Potato (Solanum tuberosum L.)
Amit, Kumar Mukesh, Vaishali, Sengar R.S., Singh Rajendra1,*, Singh S.K.2
Department of Agriculture Biotechnology , Sardar Vallabhbhai Patel University of Agriculture & Technology, Modipuram, Meerut (U.P)
1Department of Entomology , Sardar Vallabhbhai Patel University of Agriculture & Technology, Modipuram, Meerut (U.P)
2Department of Genetic & Plant Breeding, Sardar Vallabhbhai Patel University of Agriculture & Technology, Modipuram, Meerut (U.P)
*Corresponding author: Rajendra Singh, Department of Entomology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut-250110 (U.P) India. E-mail: firstname.lastname@example.org
India is the 2nd largest producer of potato in the world. Wound responsive genes are protease Inhibitors (PIs) in potato (Solanum tuberosum L.) which are the most capable weapon for developing resistance against insects by inhibiting proteases present in the gut of larvae. The potato PIs genes are a family of biotic stress resistance gene containing a PIN-I/PIN-II domain, which play crucial roles in plant development and response to biotic stresses. In this study, RNA isolation and PI gene of Kufri Himsona and its expression analysis was performed to identify PI gene members in Kufri Himsona variety. In silico Characterization of the sequence analysis by BLASTn showed 95% homology with protease inhibitor-II CM7 gene of S. tuberosum (Arran Banner, Acession no. X78275.1). Using BioEdit program, analysis of signal peptide was predicted by SignalP-4.1 and analysis of the predicted potato PI protein sequence by TargetP 1.1 server, Computation of various physical and chemical parameters by using of ProtParam Package of Expasy web server, Deduced hydrophobic index by using ProtScale package of Expasy web server, sequenced data were translated to protein sequences, and translated sequences were modelled to secondary structure prediction by PSIPRED software. Total RNA was isolated after treatment from 35 days old grow leaves of seven variety of potato by established protocols as GTC method, Trizol (Gibco), Plant RNA isolation kit (Genei) and One step RNA isolation kit (Ambion). Finally good quality of RNA was isolated from Plant RNA isolation kit (Genei). The total RNA gave two prominent bands and was used for construction of cDNA library. The expression analysis was carried out using semi quantitative RTPCR. Under stress condition in seven cultivars, where majority of the genes were upregulated after 72 h of the stress treatment. In case of artificial wounding (mechanical injury) treatment, PIs genes were up regulated with increasing duration of treatment in these cultivars as they are wound responsive genes. These results strongly suggest that isolated PI genes can be used for the development of insect resistant crop plants.