Identification of Domains-Rearranged Methyltransferases (DRM1) in Switchgrass (Panicum virgatum L.)

Stephanie O. Anyika¹, Adrianne Brown2, Venu (Kal) Kalavacharla2,3 1Department of Biological Sciences, Delaware State University, Dover, DE 19901, 2Molecular Genetics & EpiGenomics Laboratory, College of Agriculture and Related

Sciences, Delaware State University, Dover, DE 19901, 3Center for Integrated Biological and Environmental Research (CIBER), Delaware State University, Dover, DE 19901  

.  Abstract

Switchgrass (Panicum virgatum) is a native grass and under suitable soil conditions can be found throughout most of the United States except California and the Pacific Northwest. It is an excellent sustainable renewable energy crop that can be converted into cellulosic biofuels like ethanol for fuel needs. This work focuses on DNA methylation, which is an epigenetic modification that regulates key developmental and stress processes. We are analyzing DNA methyltransferases (Dnmt), which are a group of enzymes that catalyze the transfer of a methyl group to bind onto the 5’ carbon of the cytosine residue. There are four types of DNA methyltransferases; DNA methyltransferase I (MET I), Chromo-methyltransferase (CMT), Domain-Rearranged Methyltransferase (DRM) and DNA methyltransferase2 (DNMT2). DRM1 is a Dnmt that is responsible for de novo methylation which is a process that adds new methylated patterns to a DNA sequence. This research will focus on understanding the presence of DRM1expression in switchgrass under salinity stress using QPCR analysis. Our goal is to understand the role these enzymes play in de novo methylation. To determine the presence of DRM1 in switchgrass, we identified it using a plant model.

Discussion and Future Work

•  RNA was isolated from Alamo, its quantity was assessed on 1% Agarose gel based on 28S and18S ribosomal RNA fragments and quality was determined by using Nanodrop 2000 based on 260/280 ratio.

•  The cDNA was synthesized from the RNA devoid of DNA, its quality was determined on agarose gel (1%) after amplifying cDNA with AP13CTG11779 and actin primers.

Future Work •  Amplify, clone, and analyze DRM1 gene expression in switchgrass. •  Further analysis will aid in determining DRM’s function in switchgrass.

Acknowledgements We would like to thank the NIFA-funded NEWBio program, and the CIBER program at DSU. Great appreciation goes to the members of the Molecular Genetics and EpiGenomics Laboratory for their assistance.

Results

Introduction

Switchgrass (Panicum virgatum) is a perennial bunch grass averaging between three to five meters tall. This research is based on epigenetic modifications, which are changes that occur during gene expression without affecting the DNA sequence. DNA methylation is an epigenetic modification that regulates key developmental and stress process. It causes methyl groups to bind onto the 5’ carbon of the cytosine nucleotide, which is known to cause gene repression. The two types of DNA methylation are de novo and maintenance methylation. De novo methylation is a process where unmethylated cytosine residues are methylated causing newly methylated patterns to be generated by adding methyl groups to the nucleotide region. DNA methyltransferases (Dnmt) are a family of enzymes that catalyze the transfer of a methyl group to a DNA molecule. In this research we focused on Domain-Rearranged Methyltransferase (DRM1) which are proteins found only in plants. The amino acid sequences of selected DRMs have been identified in maize, tobacco, rice and many other species. Our main objective is to identify DRM1 in switchgrass.

Objective

•  Identify DRM1 in switchgrass. •  Design full-length primer DRM1 •  Amplify by conventional PCR

Materials and Methods •  Bioinformatic Resources

Ø  National Center for Biotechnology information (NCBI) Ø  Phytozome-BLAST search

•  Isolate RNA from switchgrass (Alamo) •  cDNA synthesis RT-PCR •  Gel electrophoresis •  Polymerase chain reaction (PCR)

Figure 2: Gel electrophoresis

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1- AP13CTG11779 2- Actin 3- Negative control

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References

•  Athanasia Pavlopoulou, S. K. (2007). Plant cytosine-5 DNA methyltransferases: Structure, function, and molecular evolution . Genomics , 530-541

•  Sharma, R., Singh, R., Malik, G., Deveshwar, P., Tyagi, A., Kapoor, S., & Kapoor, M. (2009). Rice cytosine DNA methyltransferases - gene expression profiling during reproductive development and abiotic stress. FEBS Journal, 6301-6311.

•  Porter, C. (1966). An Analysis of Variation Between Upland and Lowland Switchgrass, Panicum Virgatum L., in Central Oklahoma. Ecology, 980-992

Figure 3: Conventional Thermocycler

100bp Alamo Alamo 100bp Alamo Alamo 1 1a 2 2a

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor1 Steph cDNA Alamo 1 PMGG 6/25/2015 3:10:07 PM 1371.5 ng/µl 27.4 15.27 1.8 2.19 DNA 502 Steph cDNA Alamo 2 PMGG 6/25/2015 3:12:18 PM 1519.9 ng/µl 30.4 16.9 1.8 2.21 DNA 50

Figure 1: Switchgrass (Alamo)

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Table 1: cDNA concentration of switchgrass genotype (Alamo)

Figure A: Checking the Quality of Alamo RNA on a 1% Agarose gel Figure B: Used constitutive genes AP13CTG11779 and Actin primers to validate cDNA quality on a 1% Agarose gel.

28S 18S

100bp 1 2 3 100bp 1 2 3 100bp