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Improved methods for basic molecular manipulation of the red alga Porphyra umbilicalis (Rhodophyta: Bangiales)

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Abstract

The isolation of DNA and RNA from the leafy gametophyte (thallus) of red algal seaweeds such as Porphyra is still a challenge because of the high content of polysaccharides. Further molecular analysis, including restriction enzyme digestion, polymerase chain reaction (PCR), and reverse transcription (RT), could be interfered by the remaining trace amount of polysaccharides. Various protocols have been developed for molecular biological studies of Porphyra species to avoid polysaccharide contamination. Here, we compare different methods in DNA and RNA isolation from the thallus of Porphyra umbilicalis. The quality of the resulting DNA for restriction enzyme digestion and PCR and the quality of RNA for RT and RT-PCR were analyzed with PuActin3, a P. umbilicalis homolog of Porphyra yezoensis Actin3 gene, which is a potential marker for monitoring thallus maturation. Our results show that the polysaccharides could be simply, but efficiently, removed from DNA by coprecipitation with potassium acetate, and RNA could be purified from polysaccharides by a single step of lithium chloride precipitation. We successfully utilized the RNA preparation for the rapid amplification of cDNA ends to amplify the unknown upstream flanking region, including the 5′-UTR, of PuDnm1, a P. umbilicalis homolog of the Cyanidioschyzon merolae mitochondrial division gene Dnm1, and locate the putative transcription start point at −162 bp from the translation initiation site (A of the ATG codon). Our work provides a simplified basic workflow for the future molecular cloning work on the gametophyte phase of P. umbilicalis and can be adopted easily for other polysaccharide-rich seaweeds.

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Acknowledgments

This study was supported by the State Key Basic Research Project of China (#2007CB108802), the National Natural Science Foundation of China (#90817002), the Public Science and Technology Research Funds Projects of Ocean (#201105023) to S. Lu, and the National Science Foundation Research Coordination Networks (NSF 0741907, PIs, S.H. Brawley, E. Gantt, A. Grossman, J. Stiller). The isolation of the P. umbilicalis isolate P.um.1 was sponsored by the National Oceanic and Atmospheric Administration (#NA06OAR4170108, PI, S.H. Brawley). Y. Xiao is also supported by the National Science Foundation of China (#J1103512). The authors are grateful to Drs Susan Brawley and Nicolas Blouin for the help of P. umbilicalis material and to Dr Charles Yarish for valuable discussion.

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  1. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, China

    Li-En Yang, Qiu-Ping Jin, Yu Xiao & Shan Lu

  2. School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, 215500, China

    Pu Xu

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Correspondence to Shan Lu.

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Yang, LE., Jin, QP., Xiao, Y. et al. Improved methods for basic molecular manipulation of the red alga Porphyra umbilicalis (Rhodophyta: Bangiales). J Appl Phycol 25, 245–252 (2013). https://doi.org/10.1007/s10811-012-9858-5

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