Quality RNA Isolation and Efficient Construction of Rft1 (Peasy:Rft1) Gene

Abstract

Introduction-High-quality RNA isolation from a polysaccharide-rich plant such as rice for downstream application is challenging. Equally, successful gene cloning for molecular analysis is tricky as it produces negative transformants.Objective-Here we reported on qualitative and quantitative RNA, RT-PCR amplification of RFT1 gene from matured leaves of Malaysian upland rice, then construction of the gene. Methodology-The RNA was isolated from Wai cultivar leaves (indica sub-species) using three different methods, synthesized cDNA and RT-PCR amplification of RFT1 gene. A T-A cloning of the RFT1 gene into p-EASY T3 vector (pEASY:RFT1), chemical transformation into E. coliTrans1-T1 and screening of the transformed cells on selective Laix-plate, by colony PCR and RE analysis were observed.Results-The RNA integrity indicated that using guanidine isothiocyanate based method, precisely Trizol was the superlative (yield = 36.50±0.59 and purity = 2.04±0.09) for isolating quality RNA from upland rice leaves. The RT-PCR and bioinformatics analysis resulted in full-length RFT1 gene amplification; responsible for promoting flowering under LD condition. Both recombinant DNA construction and transformation were efficient with more than 50% white colonies due to deactivation of the lacZ reporter gene. Also, the gel analysis of the colony-PCR indicated an absolute RFT1 gene amplification with a singleband and double-bands (vector and insert) after RE analysis all at expected positions. Conclusion-These have shown the suitability of the RNA isolation protocols observed in producing qualityRNA for downstream applications, first ever reported on full-length RFT1 gene isolation from Malaysian upland rice and successful gene construction (pEASY:RFT1).