Sains Malaysiana 43(8)(2014): 1133–1138

Preliminary Proteomic Characterisation of Primodia and Vegetative Dikaryotic Mycelial Cells from Tiger’s Milk Mushroom (Lignosus rhinocerus)

(Kajian Awal Pencirian Proteomik Sel Primordia dan Sel Miselia Vegetatif Dikariot Cendawan Susu Harimau (Lignosus rhinocerus)

WEI HONG LAI¹*, TEIK KEE LEO², ZAMRI ZAINAL³ & FAUZI DAUD³

¹Agro-Biotechnology Institute, Ministry of Science, Technology and Innovation

c/oMalaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor,

D.E. Malaysia

²Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia

43400 UPM Serdang, Selangor, D.E. Malaysia

³School of Biosciences & Biotechnology, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, D.E. Malaysia

Received: 26 June 2013/Accepted: 17 December 2013

ABSTRACT

Tiger’s Milk mushrooms (Lignosus rhinocerus) are polypores with three distinct parts: cap (pileus), stem (stipe) and tuber (sclerotium). The stem of this medicinal mushroom is centrally connected to the brownish woody cap that grows out from the tuber underground rather than from the wood. To date, the biotic and abiotic factors that induce the growth of this mushroom are unclear and information regarding its development is scanty. Hence, the differential protein expressions of vegetative dikaryotic mycelial and primordial cells of this mushroom were investigated. Six two dimensional-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-SDS-PAGE) of 13 cm with pH3-10 containing the intracellular proteins of vegetative mycelial and primordial cells of L. rhinocerus were obtained. Analysis of 2D-SDS-PAGE using Progenesis Samespot version 4.1 yielded approximately 1000 distinct protein spots in the proteome of vegetative mycelial cells, while primordial proteome contained nearly 100 spots. Further comparison between the vegetative mycelial and primordial proteomes yielded significant up-regulation of protein expression of 5 primordial cells proteins that were labeled as P1, P2, P3, P4 and P5. These protein spots were excised, trypsin digested and submitted to mass spectrometry. Protein identification through MASCOT yielded significant identification with P1 and P2 as DnaJ domain protein, P3 and P5 as hypothetical protein while P4 as AP-2rep transcription factor. The present results suggested that P3, P4 and P5 are novel proteins that involved in the initiation of L. rhinocerus primordia. Our findings also suggested that stress response mechanism is present during fruitification of this mushroom.

Keywords: Dikaryotic mycelia; Lignosus rhinocerus; mass spectrometry; primordia; 2D-SDS-PAGE

ABSTRAK

Cendawan Susu Harimau (Lignosus rhinocerus) merupakan polipori yang terdiri daripada tiga bahagian utama iaitu payung (pileus), batang (stipe) dan umbi (sclerotium). Cendawan bernilai yang sering digunakan dalam perubatan tradisional ini mempunyai batang yang menyambungkan payung cendawan yang berwarna coklat dengan umbi yang tumbuh dari bawah tanah. Setakat ini, faktor biotik dan abiotik yang mendorong pertumbuhan cendawan ini masih tidak diketahui dan maklumat berkenaan perkembangannya adalah terhad. Maka, kajian ini dijalankan untuk mengenal pasti pengekspresan protein daripada sel vegetatif dikariot miselia dan sel primordia yang terlibat dalam pertumbuhan. Enam gel elektroforesis dua dimensi-natrium sulfat dodesil-poliakrilamid (2D-SDS-PAGE) bersaiz 13 cm dengan pH3-10 yang mengandungi protein intrasel daripada sel vegetatif dikariot miselia dan sel primordia L. rhinocerus telah berjaya diperoleh. Analisis 2D-SDS-PAGE menggunakan Progenesis Samespot (Versi 4.0) telah menghasilkan lebih kurang 1000 bintik protein daripada proteome sel vegetatif miselia manakala proteom sel primordia mengandungi lebih kurang 100 bintik protein. Perbandingan antara proteom sel vegetatif miselia dengan sel primordia mendapati terdapat lima bintik protein yang menunjukkan pengekspresan protein yang ketara daripada sel primordia yang dilabel sebagai P1, P2, P3, P4 dan P5. Bintik protein ini dipotong, dihadam dengan tripsin dan dihantar untuk analisis jisim menggunakan spektrometri jisim. Kelima-lima protein ini berjaya dikenal pasti melalui MASCOT dan P1 serta P2 dikenal pasti sebagai protein domain DnaJ, P3 serta P5 sebagai protein andaian manakala P4 sebagai protein faktor transkripsi AP-2rep. Hasil kajian mencadangkan bahawa P3, P4 dan P5 merupakan protein novel yang terlibat dalam inisiasi primordia L. rhinocerus. Di samping itu, hasil kajian juga menunjukkan kehadiran mekanisme tindak balas tekanan semasa pembuahan cendawan ini.

Kata kunci:  Dikariot miselia; Lignosus rhinocerus; primordia; spektrometri jisim; 2D-SDS-PAGE

REFERENCES

Böhmer, M., Colby, T., Böhmer, C., Bräutigam, A., Schmidt, J. & Bölker, M. 2007. Proteomic analysis of dimorphic transition in the phytopathogenic fungus Ustilago maydis. Proteomics 7: 675-685.

Bryne, C., Tainsky, M.A. & Fuchs, E. 1994. Programming gene expression in developing epidermis. Development 120: 2369-2383.

Bukau, B. & Horwich, A.L. 1998. The Hsp70 and Hsp60 chaperone machines. Cell 92: 351-366.

Chum, W.W.Y., Ng, K.T.P., Shih, R.S.M., Au, C.H. & Kwan, H.S. 2008. Gene expression studies of the dikaryotic mycelium and primordium of Lentinus edodes by serial analysis of gene expression. Mycological Research 112(8): 950-964.

Corsi, A.K. & Schekman, R. 1997. The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae. Journal of Cell Biology 137:1483-1493.

Cui, B.K., Tang, L.P. & Dai, Y.C. 2011. Morphological and molecular evidences for a new species of Lignosus (Polyporales, Basidiomycota) from tropical China. Mycologia Progress 10: 267-271.

Douanla-Meli, C. & Langer, E. 2003. A new species of Lignosus (Polyporaceae) from Cameroon. Mycotaxon 86: 389-394.

Famá, M.C., David, R., Zacchi, N., Lemos, D.R., Robinson, A.S. & Silberstein, S. 2007. The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum. Biochimica et Biophysica Acta 1773: 232-242.

Guo, C., Wong, K.H. & Cheung, P.C.K. 2011. Hot water extract of the sclerotium of Polyporus rhinocerotis Cooke enhances the immune functions of murine macrophage. International Journal of Medicinal Mushrooms 13(3): 237-244.

Huang, N.L. 1999a. Identification of the scientific name of Hurulingzhi. Acta Edulis Fungi 6: 30-32.

Huang, N.L. 1999b. A new species of medicinal mushroom- Cultivation of hurulingzhi. Edible Fungi China 18: 8-9.

Kim, Y., Nandakumar, M.P. & Marten, M.R. 2008. The state of proteome profiling in the fungal genus Aspergillus. Briefings in Functional Genomics and Proteomics 7: 87-94.

Kim, Y., Nandakumar, M.P. & Marten, M.R. 2007a. Proteome map of Aspergillus nidulans during osmoadaptation. Fungal Genetics and Biology 44: 886-895.

Kim, Y., Nandakumar, M.P. & Marten, M.R. 2007b. Proteomics of filamentous fungi. Trends in Biotechnology 25: 395-400.

Lai, W.H., Siti Murni, M.J., Fauzi, D., Abas Mazni, O. & Saleh, N.M. 2011. Optimal cultural conditions for the mycelial growth of Lignosus rhinocerus. Mycobiology 39(2): 92-95.

Lee, S.S., Chang, Y.S. & Noraswati, M.N.R. 2009. Utilization of macrofungi by some indigenous communities for food and medicine in Peninsular Malaysia. Forest Ecology and Management 257: 2062-2065.

Moser, M., Rüschof, J. & Buettner, R. 1997. Comparative analysis of AP-2a and AP-2b gene expression during murine embryogenesis. Developmental Dynamics 208: 115-124.

Moser, M., Pscherer, A., Imhof, A., Bauer, R., Kerscher, M., Amselgruber, W., Sinowatz, F., Hofstaedter, F., Schüle, R. & Buettner, R. 1995. Molecular cloning and characterization of a second AP-2 transcription activator gene, AP-2b. Development 121: 2779-2788.

Nejad, M.Y., Manesh, H.N. & Khajeh, K. 2011. Proteomics of early and late cold shock stress on thermophilic bacterium, Thermus sp. GH5. J. Proteomics 74(10): 2100-2111.

Norlidah, A., Muhammad, Z.D.H., Beng, F.L. & M. Suffian, M.A. 2013. Domestication of wild medicinal sclerotial mushroom, Lignosus rhinocerotis (Cooke) Ryden. Industrial Crops and Products 47: 256-261.

Núñez, M. & Ryvarden, L. 2001. East Asian polypores 2.Polyporaceae s. lato. Synopsis Fungorum 14: 170-522.

Oulad-Abdelghani, M., Bouillet, P., Chazaud, C., Dolle´, P. & Chambon, P. 1996. AP-2.2: A novel AP-2 related transcription factor induced by retinoic acid during differentiation of P19 embryonal carcinoma cells. Experimental Cell Research 225: 338-347.

Rudiger, S., Schneider-Mergener, J. & Bukau, B. 2001. Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone. The EMBO Journal 20:1042-1050.

Ryvarden, L. & Johansen, I. 1980. A Preliminary Polypore Flora of East Africa. Oslo: Fungi Flora. pp. 1-636.

Schorle, H., Meier, P., Buchert, M., Jaenisch, R. & Mitchell, P.J. 1996. Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381: 235-238.

Williams, T., Admon, A., Lu¨scher, B. & Tjian, R. 1998. Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes & Development 2: 1557-1569.

Zhang, J., Hagopian-Donaldson, S., Serbedzija, G., Elsemore, J., Plehn-Dujowich, D., McMahon, A.P., Flavell, R.A. & Williams, T. 1996. Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. Nature 381: 238-241.

*Corresponding author; email: weihan_1980@yahoo.com