Effect of 5' Deletions of Efficiency and Specificity of OsRGLP2 Promoter

Abstract

Germin like proteins (GLPs) are member of a large gene family and ubiquitously expressed as plant proteins. The exact mode of action and role in metabolism is not well understood. It is believed that these putative stress proteins are expressed at various developmental stages in response to abiotic and biotic stresses. The present study was designed to clone full length and 5' abbreviated Oryza sativa Root expressed GLP2 (OsRGLP2) promoter, its genetic transformation into Arabidopsis and characterization. Cloning of full length and 5' abbreviated OsRGLP2 promoters was carried out by employing GATEWAY™ Technology. Amplification was performed by specific primers designed on the promoter region. Recombinant entry clones were created by using pENTR/D-TOPO® cloning kit. Expression vectors were prepared by employing LR recombination reaction between recombinant entry clones and promoter less destination vector pHGWFS7. Confirmation was carried out by PCR and sequencing. Recombinant expression vectors were named as pNSS-F1 (Full length promoter) and pNSS-F2, pNSS-F3 and pNSS-F4 (5' deleted promoters). Plant transformation into Arabidopsis was carried out by Agrobacterium mediated floral dip method. T0 and T1 lines were established and transgenic plants were analyzed by molecular and physiological approaches. T1 transgenic lines for each promoter constructs were selected through GUS assay and tested for wound, salt and temperature stresses. Real-time PCR was performed by using two sets of primers, Actin (Housekeeping gene) and GFP (Gene of Interest). Real-time PCR analysis was done by employing 2-ΔΔCt method in which data was normalized by software inbuilt in the real-time PCR system by taking calibrator or untreated sample value xxi as 1. The graphical data shows the times fold increase or decrease in the expression with comparison with untreated control samples. Expression analysis revealed that OsRGLP2 promoter is efficient and specific to wound, salt and temperature stresses. When comparison was done between full length and 5' deleted promoters, the promoter named pNSS-F3 of 565 bp along with other two larger promoter pNSS-F1 and pNSS-F2 of sizes 1063 bp and 776 bp respectively were responding to all stresses applied during this study. However, when a further deletion was made and shortest promoter pNSS-F4 was created which comprises of 283 bp size, unable to respond against stresses applied. So it can be concluded from present study that during 5' deletion of full length OsRGLP2 promoter (creation of pNSS-F4), a critical region between P-565 and P-283 on promoter fragment was deleted which contain promoter elements necessary to respond against certain stresses like wound stress, salt stress and temperature stress. While during other deletions (in case of pNSS-F2 and pNSS-F3) in which when the critical part was intact, the promoter responded to applied stresses. It can be further concluded that pNSS-F3 is a good replacement for full length promoter. Due to smaller size of pNSS-F3 promoter (565 bp) and it‟s efficient and specific response against abiotic stresses it can be fused with other promoter and used in combination against certain abiotic or biotic stresses.