Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing

Angelina Angelova, Sang Hycuk Park, John Kyndt, Kevin Fitzsimmons, Judith K. Brown

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

Original languageEnglish (US)
Pages (from-to)209-218
Number of pages10
JournalJournal of Applied Phycology
Volume26
Issue number1
DOIs
StatePublished - Feb 14 2014

Keywords

  • Chloroplast isolation
  • Oleaginous algae
  • Sonication

ASJC Scopus subject areas

  • Aquatic Science
  • Plant Science

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