HOT PHENOL EXTRACTION OF TOTAL RNA FROM Thermoascus aurantiacus AND CHARACTERIZATION OF ITS THERMOSTABLE XYLANASE GENE
Abstract
Total RNA was successfully isolated using hot phenol extraction method. Three bands representing the 18S, 5.8S and 28S rRNA was observed. No heavy smearing was observed in the RNA band patterns, indicating low levels of polysaccharide contamination, when subjected to 1% agarose gel electrophoresis. Genomic DNA was eliminated using DNase I digestion and lithium chloride (LiCl) precipitation. Two-steps reverse transcriptase polymerase chain reaction (RT-PCR) using M-MuLV Reverse Transcriptase and sequence specific primers for xylanase gene, XynA(F) and XynA(R), successfully generated the target amplicon of 500 base pairs (bp). Sequence analysis of the PCR product indicated as partial sequence of Thermoascus aurantiacus xylanase gene (XynA) deposited in the NCBI GenBank with accession number: AF127529.1 and AJ132635.1. Hot phenol extraction is useful for extracting large quantities of total RNA sufficient for complementary DNA (cDNA) synthesis in shorter period of time.References
Damaso, M.C.T., Almeida, M.S., Kurtenbach, E., Martins, O.B., Pereira Jr., N., Andread, M.M.C., & Albano, R.M. (2003). Optimized expression of a thermostable xylanase from Thermomyces lanuginosus in Pichia pastoris. Applied Environmental Microbiology, 69(10): 6064-6072.
https://doi.org/10.1128/AEM.69.10.6064-6072.2003
Emami, K. & Hack, E. (2000). Characterization of a xylanase gene from Cochliobolus sativus and its expression. Journal of Mycological Research, 105(3):352 - 359.
https://doi.org/10.1017/S0953756201003689
Gupta, S., Bhushan, B. & Hoondal, G.S. (2000). Isolation, purification and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. Journal of Applied Microbiology, 88:325 - 334.
https://doi.org/10.1046/j.1365-2672.2000.00974.x
Maheshwari, R., Bharadwaj, G. & Bhat, M.K. (2000). Thermophilic fungi: their physiology and enzymes. Microbiology and Molecular Biology Reviews, 64(3):461 - 488.
https://doi.org/10.1128/MMBR.64.3.461-488.2000
Nel, S. (2001). Cloning of the XynA gene from Thermomyces lanuginosus and expression in Saccharomyces cerevisiae MSc. Thesis. Faculty of Natural and Agricultural Sciences, Department of Microbiology and Biochemistry University of the Free State Bloemfontein South Africa.
Saarelainen, R., Paloheimo, M., Fagerstrom, R., Suominen, P.L. & Nevalainen, K.M.H. (1993). Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2. Molecular and General Genetics, 241:497-503.
https://doi.org/10.1007/BF00279891
Shibuya, H., Kaneko, S. & Hayashi, K. (2000). Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling. Journal of Biochemistry, 349:651 - 656.
Copyright (c) 2016 Borneo Journal of Resource Science and Technology
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Copyright Transfer Statement for Journal
1) In signing this statement, the author(s) grant UNIMAS Publisher an exclusive license to publish their original research papers. The author(s) also grant UNIMAS Publisher permission to reproduce, recreate, translate, extract or summarize, and to distribute and display in any forms, formats, and media. The author(s) can reuse their papers in their future printed work without first requiring permission from UNIMAS Publisher, provided that the author(s) acknowledge and reference publication in the Journal.
2) For open access articles, the author(s) agree that their articles published under UNIMAS Publisher are distributed under the terms of the CC-BY-NC-SA (Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License) which permits unrestricted use, distribution, and reproduction in any medium, for non-commercial purposes, provided the original work of the author(s) is properly cited.
3) For subscription articles, the author(s) agree that UNIMAS Publisher holds copyright, or an exclusive license to publish. Readers or users may view, download, print, and copy the content, for academic purposes, subject to the following conditions of use: (a) any reuse of materials is subject to permission from UNIMAS Publisher; (b) archived materials may only be used for academic research; (c) archived materials may not be used for commercial purposes, which include but not limited to monetary compensation by means of sale, resale, license, transfer of copyright, loan, etc.; and (d) archived materials may not be re-published in any part, either in print or online.
4) The author(s) is/are responsible to ensure his or her or their submitted work is original and does not infringe any existing copyright, trademark, patent, statutory right, or propriety right of others. Corresponding author(s) has (have) obtained permission from all co-authors prior to submission to the journal. Upon submission of the manuscript, the author(s) agree that no similar work has been or will be submitted or published elsewhere in any language. If submitted manuscript includes materials from others, the authors have obtained the permission from the copyright owners.
5) In signing this statement, the author(s) declare(s) that the researches in which they have conducted are in compliance with the current laws of the respective country and UNIMAS Journal Publication Ethics Policy. Any experimentation or research involving human or the use of animal samples must obtain approval from Human or Animal Ethics Committee in their respective institutions. The author(s) agree and understand that UNIMAS Publisher is not responsible for any compensational claims or failure caused by the author(s) in fulfilling the above-mentioned requirements. The author(s) must accept the responsibility for releasing their materials upon request by Chief Editor or UNIMAS Publisher.
6) The author(s) should have participated sufficiently in the work and ensured the appropriateness of the content of the article. The author(s) should also agree that he or she has no commercial attachments (e.g. patent or license arrangement, equity interest, consultancies, etc.) that might pose any conflict of interest with the submitted manuscript. The author(s) also agree to make any relevant materials and data available upon request by the editor or UNIMAS Publisher.
