Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

Abstract

Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Deposition of lignin in four species of Saccharum

“…RT-PCR reactions were carried out in a thermal cycler (Veriti 96-Well Thermal Cycler-AB Applied Biosystems) following the parameters of Llerena et al . 112 . The amplification products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide and observed by a photo-documenter Gel Doc 2000 (Biorad).…”