TRANSFORMASI GENETIK DAN EKSPRESI MUTAN SUCROSE PHOSPHATE SYNTHASE PADA TANAMAN TOMAT
Main Article Content
Abstract
Sucrose phosphate synthase (SPS) merupakan enzim kunci yang bertanggung jawab dalam sintesis sukrosa. Dalam regulasinya, aktifitas SPS dipengaruhi oleh alosterik efektor glukosa-6-fosfat (G6P) yang diduga dapat berikatan pada domain N-terminus SPS. Untuk mengetahui peran N-terminus pada regulasi SPS, dilakukan mutasi SPS dengan penghilangan domain N-terminus (∆N-SPS). Gen ∆N-SPS diinsersi pada tanaman tomat melalui transformasi genetik dengan efisiensi transformasi 5%. Tiga tanaman transgenik tomat (event4.20; 5.5.1; dan 5.10) didapatkan dan positif terkonfirmasi melalui analisis PCR. Ekspresi mutan dikarakterisasi melalui analisis enzimatik. Hasil menunjukkan bahwa tanaman tomat transgenik ∆N-SPS tidak dipengaruhi regulasi alosterik G6P dan aktifitas SPS 2 kali lipat lebih tinggi daripada tanaman bukan transgenik. Ini menunjukkan bahwa SPS dengan delesi domain N-terminus dapat terekspresi aktif pada tanaman.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Authors who publish with this journal agree to the following terms:
a). Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Attribution-NonCommercial-ShareAlike 4.0 International that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
b). Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
c). Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
References
Apriasti R, Widyaningrum S, Hidayati WN, Sawitri WD, Darsono N, Hase T, Sugiharto B (2018) Full sequence of the coat protein gene is required for the induction of pathogen-derived resistance against sugarcane mosaic virus in transgenic sugarcane. Mol Biol Rep 45(6):2749–2758. doi: 10.1007/s11033-018-4326-1
Baskaran S, Roach PJ, DePaoli-Roach, AA, Hurley TD (2010) Structural basis for glucose-6-phosphate activation of glycogen synthase. Proc Natl Acad Sci U S A 107(41):17563-17568. doi: 10.1073/pnas.1006340107
Breyer D, Kepertekh L, Reheul D (2014) Alternatives to antibiotic resistance marker genes for in vitro selection of genetically modified plants – scientific developments, current use, operational access, and biosafety considerations. CRC Crit Rev Plant Sci 33(4):286-330. Doi: 10.1080/07352689.2013.870422
But SY, Khmelenina VN, Reshetnikov AS, TrotsenkoYA (2013) Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT. FEMS Microbiol Lett 347(1):43-51. doi: 10.1111/1574-6968.12219
Coleman HD, Beamish L, Reid A, Park JY, Mansfield SD (2010) Altered sucrose metabolism impacts plant biomass production and flower development. Transgenic Res 19(2):269-283. doi: 10.1007/s11248-009-9309-5
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1:19–21. https ://doi.org/10.1007/BF027 12670
Diricks M, De Bruyn F, Van Daele P, Walmagh M, Desmet T (2015) Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes. Appl Microbiol Biotechnol 99(20), 8465–8474. doi: 10.1007/s00253-015-6548-7
Huber SC, Kerr PS, and Rufty TW (1985) Diurnal changes in sucrose phosphate synthase activity in leaves. Physiol Plant 64:81-87
Huber SC, Huber JL (1996) Role and regulation of sucrose-phosphate synthase in higher plants. Annu Rev Plant Physiol Plant Mol Biol 47:431-444. doi: 10.1146/annurev.arplant.47.1.431
Lemoine R, Camera SL, Atanassova R, Dedaldechamp F, Allario T, Pourtau N, Bonnemain JL, Laloi M, Thevenot PC, Mauorousset L, Faucher M, Girousse C, Lemonnier P, Parrilla J and Durand M (2013) Source-to-sink transport of sugar and regulation by environmental factors. Front Plant Sci 4(272):1-21. doi: 10.3389/fpls.2013.00272
Maloney VJ, Park JY, Unda F, Mansfield SD (2015) Sucrose phosphate synthase and sucrose phosphate phosphatase interact in planta and promote plant growth and biomass accumulation. J Exp Bot 66(14): 4383-4394. doi: 10.1093/jxb/erv101
Neliana IR, Sawitri WD, Ermawati N, Handoyo T, Sugiharto B (2019) Development of allergenicity and toxicity assessment methods for evaluating transgenic sugarcane overexpressing sucrose-phosphate synthase. Agronomy 9(1):23. doi: 10.3390/agronomy9010023
Ruan YL (2012) Signaling role of sucrose metabolism in development. Mol Plant 5(4):763-765. doi: 10.1093/mp/sss046
Sawitri WD, Narita H, Ishizaka-Ikeda E, Sugiharto B, Hase T, Nakagawa A (2016) Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: An importance of the N-terminal domain for an allosteric regulatory property. J Biochem 159(6): 599–607. doi: 10.1093/jb/mvw004
Sawitri WD, Sugiharto B (2018a) Rekayasa Sucrose Phosphate Synthase untuk Meningkatkan Sukrosa Sebagai Sumber Karbon dan Energi Bagi Pertumbuhan Tanaman. In: Wikantika K (ed) Bunga Rampai ForMIND 2018. ITB Press, Bandung, Indonesia, pp 165-172. ISBN: 978-602-0705-19-4
Sawitri WD, Afidah SN, Nakagawa A, Hase T, Sugiharto B (2018b). Identification of UDP-glucose binding site in glycosyltransferase domain of sucrose phosphate synthase from sugarcane (Saccharum officinarum) by structure-based site-directed mutagenesis. Biophys Rev 10(2):293–298. doi: 10.1007/s12551-017-0360-9
Seger M, Gebril S, Tabilona J, Peel A, Sengupta-Gopalan, C (2014) Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco. Planta 241(1):69-81. doi: 10.1007/ s00425-014-2165-4
Solis-Guzman MG, Arguello AG, Lopez BJ, Ruiz HLF, Lopez MJE, Sanchez CL, Carreon AY, Martinez TM (2017) Arabidobsis thaliana sucrose phosphate synthase (sps) genes are expressed differentially in organ and tissue, and their transcription is regulated by osmotic stress. Gene expr patterns 25-26:92-101.doi: 10.1016/j.gep.2017.06.001
Sugiharto B, Sakakibara H, Sumadi, Sugiyama T (1997). Differential expression of two genes for sucrose-phosphate synthase in sugarcane: Molecular cloning of the cDNAs and comparative analysis of gene expression. Plant Cell Physiol 38(8):961-965. doi: 10.1093/ oxfordjournals.pcp.a029258
Sugiharto B (2018) Chapter 8: Biotechnology of drought-tolerant sugarcane. In: Oliveira AD (ed) Sugarcane Technology and Research. IntechOpen, Florida, USA, pp 139-165. doi: 10.5772/intechopen.69564
Sun S, Kang XP, Xing XJ, Xu, XY, Cheng J, Zheng SW, Xing GM (2015) Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv. Hezuo 908) with improved efficiency. Biotechnol Biotechnol Equip 29(5):861-868 doi: 10.1080/13102818.2015.1056753
Toroser D, McMichael Jr R, Krause KP, Kurrect J, Sonnewald U, Stitt, Hubber SC (1999) Site-directed mutagenesis of serine-158 demonstrated its role in spinach leaf sucrose-phosphate synthase modulation. Plant J 17(4):407-413. doi: 10.1046/j.1365-313X.1999.00389.x
Torres WK, Kerr PS, Huber SC (1987) Isolation and characterization of multiple form of maize leaf sucrose-phosphate synthase. Physiol Plantarium 70:653-658
Verma AK, Upadhyay SK, Verma PC, Solomon S, Singh SB (2011) Functional analysis of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in sugarcane (Saccharum) cultivars. Plant Biol 13(2):325-332. doi: 10.1111/j.1438-8677.2010.00379.x
Volkert K, Debast S, Voll LM, Schieβl I, Hofmann J, Schneider S, Börnke F (2014) Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis. J Exp Bot 65(18):5217-5229. doi: 10.1093/jxb/eru282
Wang J, Du J, Mu X, Wang P (2017) Cloning and characterization of the Cerasus humilis sucrose phosphate synthase gene (ChSPS1). Plos One 12(10): e0186650. doi: 10.1371/journal.pone.0186650
Wang D, Zhao J, Hu B, Li J, Qin Y, Chen L, Qin Y, Hu G (2018) Identification and expression profile analysis of the sucrose phosphate synthase gene family in Litchi chinensis Sonn. PeerJ 6:e4379. doi: 10.7717/peerj.4379
Yang L, Wang C, Wang L, Xu C, Chen K (2013) An efficient multiplex PCR assay for early detection of Agrobacterium tumifaciens in transgenic plant material. Turk J Agric For 37:157-162. doi: 10.3906/tar-1009-1265
Yonekura M, Naohiro A, Hirose T, Onai K, Ishiura M, Okamura M, Ohsugi R, Ohto C (2013) The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose. Front Plant Sci 4(31):1-8. doi: 10.3389/fpls.2013.00031