New efficient DNA extraction method to access the microbiome of Ricinus communis seeds

Abstract

ABSTRACT. Ricinus communis (castor bean) seeds are used to produce an alcohol-soluble oil that is used in more than 400 industrial processes. Despite its economic importance, there has been little research on the endophytic microbiota of castor bean seeds. This microbiota is important for plant metabolic processes and may have considerable biotechnological potential, such as production of lipases and plant growth promoter agents. We evaluated several DNA extraction methodologies in order to access the microbial diversity of castor bean through a metagenomic approach. Based on our observations, we developed a new methodology that takes advantage of the low solubility of calcium phosphates and the high affinity of these phosphates for proteins and polysaccharides. The extracted DNA quality was evaluated by PCR, using a selective primer pair for bacterial and mitochondrial 16S rDNA genes (799F and 1492R). We found this methodology quantitatively and qualitatively more efficient than the other approaches. In evaluating this new extraction methodology, we found that the difficulties of DNA extraction from castor bean seeds, such as abundant oil, polysaccharides, phenolic compounds, and plant enzymes, could be overcome. The resulting extracts had high concentration and purity, ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (3): 3128-3135 (2013) New efficient DNA extraction to access oil seed microbiome 3129 and they were obtained faster than with previous methods. The samples contained virtually all of the DNA, including the microbial DNA; this was validated by PCR analysis.

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“…To perform direct DNA extraction from the enrichment broths: LS; PW and BHI with 3% NaCl, it was used a method from the phenol‐chloroform extraction described by Santos Jr. et al . (). The extracted DNA was purified using Qiaquick PCR Purification Kit (Quiagen, Hilden, Germany) before the PCR technique which was performed to identify the specific genes described earlier.…”

Development of an efficient chromatin immunoprecipitation method to investigate protein-DNA interaction in oleaginous castor bean seeds

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