Sains Malaysiana 54(3)(2025): 769-783

http://doi.org/10.17576/jsm-2025-5403-13

 

Identification and In-Silico Analysis of a Cellulose Synthase-Like Gene on Eddoe Taro

 (Pengenalpastian dan Analisis In-Silico Gen Selulosa Seperti Sintase pada Eddoe Taro)

 

DELVI MARETTA^1,*, IS HELIANTI², DEVIT PURWOKO³, EDI SANTOSA⁴ & SOBIR⁴

 

¹Research Center for Horticulture, National Research and Innovation Agency, Republic of Indonesia (BRIN), Cibinong Science Center, Jl. Raya Bogor KM 46 Cibinong, West Java 16915, Indonesia

²Research Center for Genetic Engineering, National Research and Innovation Agency, Republic of Indonesia (BRIN), Cibinong Science Center, Jl. Raya Bogor KM 46 Cibinong, West Java 16915, Indonesia

³Research Center for Applied Botany, National Research and Innovation Agency, Republic of Indonesia (BRIN), Cibinong Science Center, Jl. Raya Bogor KM 46 Cibinong, West Java 16915, Indonesia

⁴Department Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, Jl Meranti Kampus IPB Darmaga, Bogor, West Java 16680, Indonesia

 

Received: 14 May 2024/Accepted: 16 December 2024

 

Abstract

Glucomannan is a non-starch carbohydrate predominantly found in tubers, serving as a significant resource for food and health industries. Despite the widely recognized glucomannan content in taro tubers, there is limited understanding regarding the gene level. Therefore, this research aimed to identify putative Cellulose Synthase-like A (CslA) gene sequences associated with glucomannan biosynthesis in eddoe taro plant. Genome isolation was carried out on six genotypes of eddoe taro, each showing different glucomannan content. A pair of primers designed from the mannan synthase encoding gene sequences obtained from the NCBI. Subsequently, sequences of the PCR product were analyzed for identification and in-silico analysis. The result of in-silico RFLP analysis showed that six genotypes had polymorphic allelic fragments. The DNA sequences showed a high similarity to CslA gene, among representative taro tubers compared to the reference plants. A total of three nucleotide sequences fragments from the S7 and S34 genotypes as well as two from S15, S28, S30, and S36, corresponded to CslA gene of Amorphophallus konjac. Phylogenetic analysis based on nucleotide sequences showed that S7 and S34 had distinctive characteristics, indicating specific and wide adaptation, respectively. Despite the presence of single nucleotide polymorphism, the in-silico transcription-translation showed that the protein constructed had a highly similar consensus motif. These results suggested the identified sequences as a potential CslA-encoding gene that had functioned in the biosynthesis of mannan synthase to produce glucomannan in taro plants.

Keywords: Biosynthesis; CslA gene; glucomannan; sequences polymorphism; taro breeding

 

Abstrak

Glukomanan ialah karbohidrat bukan-kanji dalam ubi dan digunakan secara meluas dalam industri makanan dan kesihatan. Ubi keladi diketahui mengandungi glukomanan, namun pengetahuan berkaitan perkara ini terutamanya di tahap gen masih sedikit. Tujuan penyelidikan ini untuk mengenal pasti jujukan gen selulosa seperti sintase A (CslA) yang mungkin memainkan peranan dalam biosintesis glukomanan pada keladi jenis eddoe. Pengasingan genom telah dijalankan pada enam genotip keladi eddoe yang mempunyai perbezaan yang ketara pada kandungan glukomanan. Sepasang primer yang direka daripada urutan gen pengekodan manan sintase yang diperoleh daripada bank gen NCBI. Jujukan produk PCR dianalisis untuk pengenalpastian dan analisis in silico. Hasil analisis in silico RFLP bagi jujukan menunjukkan bahawa enam genotip mempunyai serpihan alel polimorfik. Analisis jujukan DNA menunjukkan persamaan yang tinggi dengan gen CslA, antara keladi perwakilan berbanding dengan tumbuhan rujukan. Terdapat tiga serpihan jujukan nukleotida daripada genotip S7 dan S34 dan dua serpihan daripada S15, S28, S30 dan S36 yang sepadan dengan gen CslA Amorphophallus konjac. Analisis filogenetik berdasarkan jujukan nukleotida menunjukkan bahawa S7 dan S34 dipisahkan daripada genotip lain. Genotip S34 telah dikenal pasti sebagai adaptasi sekitaran luas manakala S7 mempunyai adaptasi lokasi khusus. Penjajaran menemui polimorfisme nukleotida tunggal tetapi terjemahan transkripsi in silico mendedahkan penjajaran protein dengan motif konsensus serupa yang tinggi. Ia dianggap bahawa jujukan yang ditemui ialah gen pengekodan CslA yang berpotensi yang telah berfungsi dalam biosintesis manan sintase untuk menghasilkan glukomanan dalam keladi.

Kata kunci: Biosintesis; gen CslA; glukomanan; pembiakan talas; polimorfisme jujukan

 

REFERENCES

Alonso-Sande, M., Teijeiro-Osorio, D., Remuñán-López, C. & Alonso, M.J. 2009. Glucomannan, a promising polysaccharide for biopharmaceutical purposes. Journal of Pharmaceutics and Biopharmaceutics 72(2): 453-462. doi:10.1016/j.ejpb.2008.02.005

Aulia, N., Putri, C.K., Khairiyah, L., Khairunnisa, F.A. & Achyar, A. 2021. Analysis of genetic variation of measles virus genotype and isolate M-F intergenic spacer sequences PopSet: 2105287799 using Restriction Fragment Length Polymorphism (RFLP) in-silico. Prosiding Seminar Nasional Biologi 1(2): 1139-1146. https://semnas.biologi.fmipa.unp.ac.id/index.php/prosiding/article/view/298/224

Baucke, L., Miele, E. & Staiano, A. 2004. Fiber (glucomannan) is beneficial in the treatment of childhood constipation. Pediatrics 113(3): e259-e264. doi:10.1542/peds.113.3.e259

Birketvedt, G.S., Shimshi, M., Erling, T. & Florholmen, J. 2005. Experiences with three different fiber supplements in weight reduction. Medicinal Science Monitoring 11(1): PI5-8.

Chairul, C. & Chairul, S.M. 2006. Isolasi glukomanan dari dua jenis araceae: Talas (Colocasia esculenta (L.) Schott) dan ıles-ıles (Amorphophallus campanulatus Blumei). Berita Bio 8(3): 171-178.

Chauhan, A., Modgil, M., Rajam, M.V.R., Sharma, J.N. & Siddappa, S. 2023. Isolation, cloning and in-silico analysis of β-tubulin gene from apple leaf blotch fungus Marssonina coronaria. Indian Phytopathology 76: 371-382. https://doi.org/10.1007/s42360-023-00614-z

Gao, Y., Zhang, Y., Feng, C., Chu, H., Feng, C., Wang, H., Wu, L., Yin, S., Liu, C., Chen, H., Li, Z., Zou, Z. & Tang, L. 2022. A chromosome-level genome assembly of Amorphophallus konjac provides insights into konjac glucomannan biosynthesis. Computational and Structural Biotechnology Journal 20: 1002-1011. doi:10.1016/j.csbj.2022.02.009

Gao, Y., Yin, S., Yang, H., Wu, L. & Yan, Y. 2017. Genetic diversity and phylogenetic relationships of seven Amorphophallus species in southwestern China revealed by chloroplast DNA sequences. Mitochondrial DNA Part A 29(5): 679-686. doi:10.1080/24701394.2017.1350855

Gille, S., Cheng, K., Skinner, M.E., Liepman, A.H., Wilkerson, C.G. & Pauly, M. 2011. Deep sequencing of voodoo lily (Amorphophallus konjac): An approach to identify relevant genes involved in the synthesis of the hemicellulose glucomannan. Planta 234(3): 515-526. doi:10.1007/s00425-011-1422-z

Goubet, F., Barton, C.J., Mortimer, J.C., Yu, X., Zhang, Z., Miles, G.P., Richens, J., Liepman, A.H., Seffen, K. & Dupree, P. 2009. Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis. The Plant Journal 60(3): 527-538. doi:10.1111/j.1365-313X.2009.03977.x

Hall, T.A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series. pp. 95-98.

Hu, H., Liu, J., Wang, B., An, J. & Wang, Q. 2019. Characterization of the complete chloroplast genome of Amorphophallus konjac (Araceae) and its phylogenetic analysis. Mitochondrial DNA Part B 4(1): 1658-1659. doi:10.1080/23802359.2019.1606683

Jo, J., Kang, M.Y., Kim, K.S., Youk, H.R., Shim, E.J., Kim, H., Park, J.S., Sim, S.C., Yu, B.C. & Jung, J.K. 2022. Genome‑wide analysis‑based single nucleotidepolymorphism marker sets to identify diverse genotypes in cabbage cultivars (Brassica oleracea var. capitata). Scientific Reports 12: 20030. https://doi.org/10.1038/s41598-022-24477-y

Karima, R., Elya, B. & Sauriasari, R. 2023.  Mechanism of action of glucomannan as a potential therapeutic agent for type 2 diabetes mellitus.  Tropical Journal of Natural Product Research 7(12): 5460-5469. DOI 10.26538/tjnpr/v7i12.15

Keithley, J. & Swanson, B. 2005. Glucomannan and obesity: A critical review. Alternative Therapies in Health and Medicine 11(6): 30-34.

Kelley, L.A. & Sternberg, M.J.E. 2009. Protein structure prediction on the web: A case study using the Phyre server. Nature Protocols 4(3): 363-371.

Krieger, E., Koraimann, G. & Vriend, G. 2002. Increasing the precision of comparative models with YASARA NOVA- A selfparameterizing force field. Proteins: Structure, Function and Genetics 47(3): 393-402.

Liepman, A.H., Wilkerson, C.G. & Keegstra, K. 2005. Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases. Proceedings of the National Academy of Sciences of the United States of America 102(6): 2221-2226. doi:10.1073/pnas.0409179102

Liepman, A.H., Nairn, C.J., Willats, W.G.T., Sorensen, I., Roberts, A.W. & Keegstra, K. 2007. Functional genomic analysis supports conservation of function among cellulose synthase-like a gene family members and suggests diverse roles of mannans in plants. Plant Physiology 143(4): 1881-1893. doi:10.1104/pp.106.093989

Mahendhiran, M., Ramirez, P.J.H., Escobedo, G., Medrano, R.M., Canto, C.B., Tzec-Simá, M., Grijalva, A.R. & James, K.A. 2014. Single nucleotide polymorphisms in partial sequences of the gene encoding the large sub-units of ADP-glucose pyrophosphorylase within a representative collection of 10 Musa genotypes. Electronic Journal of Biotechnology 17(3): 137-147. doi:10.1016/j.ejbt.2014.04.004

Marchler-Bauer, A., Zheng, C., Chitsaz, F., Derbyshire, M.K., Geer, L.Y., Geer, R.C., Gonzales, N.R., Gwadz, M., Hurwitz, D.I., Lanczycki, C.J., Lu, F., Lu, S., Marchler, G.H., Song, J.S., Thanki, N., Yamashita, R.A., Zhang, D. & Bryant, S.H. 2013. CDD: Conserved domains and protein three-dimensional structure. Nucleic Acids Research 41(Database issue): D348-D352. https://doi.org/10.1093/nar/gks1243 

Maretta, D., Sobir, Helianti, I., Ridwan, D., Purwono & Santosa, E. 2023. Glucomannan content stability of Eddoe taro tuber based on parametric, non parametric, and AMMI analysis. Sains Malaysiana 52(11): 3135-3145.  http://doi.org/10.17576/jsm-2023-5211-09

Maretta, D., Sobir, Helianti, I., Purwono & Santosa, E. 2020. Genetic diversity in eddoe taro (Colocasia esculenta var. Antiquorum) from Indonesia based on morphological and nutritional characteristics. Biodiversitas 21(8): 3525-3533. https://doi.org/10.13057/biodiv/d210814

Mashudi, S., Putri, D.R. & Hariyatmi, Aziz, Y.S. 2023. The impact of konjac glucomannan in reducing blood glucose levels. Community Practitioner 20(10): 387-393. DOI: 10.5281/zenodo.10060277

Moreira, L.R.S. & Filho, E.X.F. 2008. An overview of mannan structure and mannan degrading enzyme systems. Applied Microbiology and Biotechnology 79: 165-178.

Suzuki, S., Li, L., Sun, Y.H. & Chiang, V.L. 2006. The cellulose synthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpa. Plant Physiology 142(3): 1233-1245. doi:10.1104/pp.106.086678

Tasma, I.M. 2015. Utilization of genome sequencing technology to accelerate plant breeding program. Jurnal Penelitian dan Pengembangan Pertanian 32(2): 159-168.

Theodore, G.S. 2000. Mitochondrial DNA and the peopling of the new world genetic variations among native Americans provide further clues to who first populated the Americas and when they arrived. American Scientist Online https://www.sas.upenn.edu/~tgschurr/pdf/Am%20Sci%20Article%202000.pdf

Thompson, J.D., Gibson, T.J. & Higgins, D.G. 2003. Multiple sequence alignment using ClustalW and ClustalX. Current Protocols in Bioinformatics Chapter 2:Unit 2.3.

Wigoeno, Y.A., Azrianingsih, R. & Roosdiana, A. 2013. Analisis kadar glukomanan pada umbi porang (Amorphophallus muelleri Blume) menggunakan refluks kondensor. Jurnal Biotropika 1(5): 231-235.

Yadav, S., Yadava, Y.K., Kohli, D., Meena, S., Kalwan, G., Bharadwaj, C., Gaikwad, K., Arora, A. & Jain, P.K. 2022. Genome‑wide identification, in-silico characterization and expression analysis of the RNA helicase gene family in chickpea (C. arietinum L.). Scientific Reports 12: 9778. https://doi.org/10.1038/s41598-022-13823-9

Yang, D., Yuan, Y., Wang, L., Wang, X., Mu, R., Pang, J., Xiao, J. & Zheng, Y. 2017. A review on konjac glucomannan gels: Microstructure and application. International Journal of Molecular Sciences 18(11): 2250. doi:10.3390/ijms18112250

Yeriska, F., Umar, M.Z., Hijriah, N.W., Fadhlurrohman, R. & Achyar, A. 2021. Analisis variasi genetik sekuen gen pengkode protein spike virus MERS-CoV (PopSet: 1843801421) menggunakan RFLP secara in-silico. Prosiding Seminar Nasional Biologi 1(2): 898-906. https://semnas.biologi.fmipa.unp.ac.id/index.php/prosiding/article/view/145?articlesBySameAuthorPage=3

Zalewski, B.M., Chmielewska, A. & Szajewska, H. 2015. The effect of glucomannan on body weight in overweight or obese children and adults: A systematic review of randomized controlled trials. Nutrition 31(3): 437-442. https://doi.org/10.1016/j.nut.2014.09.004

 

*Corresponding author; email: delvi.maretta@brin.go.id