Peningkatan biosintesis sukrosa tanaman tebu (Saccharum officinarum L.) melalui over ekspresi gen Sucrose Phosphate Synthase (SPS)

MISWAR, Promotor Prof.Dr. Sukarti Moeljopawiro, M.App.Sc

2007 | Disertasi | S3 Ilmu Bioteknologi

Dalam tanaman sucrose phosphate synthase (SPS) memegang peranan penting dalam sintesis sukrosa dari UDP-glucose dan fructose-6-P. Enzim ini menentukan kemampuan tanaman untuk mesintesis dan mengakumulasikan sukrosa serta mempengaruhi alokasi senyawa karbon yang diasimilasi. Tujuan penelitian ini adalah untuk meningkatkan kemampuan tebu mensintesis sukrosa dengan over-ekspresi gen SoSPS1 yang ditransformasikan menggunakan Agrobacterium tumefaciens. Sebelum digunakan untuk transformasi ke tanaman tebu, gen SoSPS1 diekspresikan pada E. coli dan tanaman tembakau sebagai model. E. coli BL 21 ditransformasi dengan gen SoSPS1 yang dikonstruksi pada plasmid pTrc99. Gen SoSPS1 dikonstruksi pada pBI121 dibawah kontrol promoter CaMV 35S (pKYS) dan ditransformasikan ke dalam Agrobacterium tumefaciens LBA 4404 (Agrobacterium::pKYS). Potongan daun tembakau dan potongan daun tebu cv R579 yang masih menggulung diinfeksi dengan Agrobacterium::pKYS, ditumbuhkan pada media agar sampai menjadi tanaman baru. Tanaman tembakau dan tebu baru yang didapat ditanam pada media tanah dan kemudian dianalisis. E. coli transforman dapat mengekspresikan gen SoSPS1 sampai tingkat translasi dan menghasilkan protein SoSPS1 aktif. Berdasarkan analisis PCR dan western blot didapat 3 tembakau transforman (T.3, T.13, T.17) yang positif mengandung transgen SoSPS1. Aktivitas SPS pada ketiga transforman tersebut meningkat masing-masing sebesar 114,96%; 165,76% dan 118,49% dibandingkan kontrol. Kandungan sukrosa daun tembakau T.3 dan T.13 meningkat masingmasing sebesar 118,49% dan 174,69%, sedang pada T.17 turun menjadi 93,64%. Berdasarkan hasil analisis PCR terhadap tebu hasil transformasi diperoleh 4 transforman (T.4, T.5, T.6, T.7) yang positif mengandung gen SoSPS1 yang dikendalikan oleh promoter CaMV. Analisis western blot menunjukkan bahwa SPS transgen dapat diekspresikan pada tingkat translasi dan menghasilkan jumlah protein SPS yang lebih tinggi dibandingkan kontrol. Over-ekspresi gen SoSPS1 menghasilkan peningkatan aktivitas SPS daun tebu T.4, T.5, T.6 dan T.7 masing-masing 140,85%; 228,17%; 173,94% dan 280,28% dan kandungan sukrosanya meningkat masing-masing 216,28%; 193,02%; 188,37% dan 176,74% demikian pula dengan rasio antara sukrosa:pati daun. Peningkatan aktivitas SPS daun tebu transforman diikuti oleh peningkatan aktivitas enzim pemecah sukrosa, terutama SS dan AI. Tanaman tebu transforman K1 dan B1 yang mengekspresikan xviii gen SoSPS1 mempunyai kandungan sukrosa batang lebih tinggi dibandingkan tebu kontrol. Pada tebu transforman generasi pertama, K1 dan B1, aktivitas SPS yang terlalu tinggi menyebabkan penurunan kandungan sukrosa daun.

Sucrose phosphate synthase (SPS) plays a crucial role in the synthesis of sucrose from UDP-glucose and fructose-6-P in plant. This enzyme determines the ability of plant to synthesize and accumulate sucrose, and also affect partitioning of carbon assimilate. This experiment was carried out to increase the ability of sugarcane to synthesize of sucrose through over-expression of SoSPS1 gene that transformed using Agrobacterium tumefaciens. Prior to be used for transformation into sugarcane, the expression of SoSPS1 gene were assayed in E. coli and tobacco as model plant. E. coli BL21 transformed with pTrc99A plasmid that contained SoSPS1 gene. SoSPS1 gene was constructed in pBI121 under the control of the promoter of CaMV 35S (pKYS), and transformed into Agrobacterium tumefaciens LBA 4404 (Agrobacterium::pKYS). The pieces of tobacco leaf and spindle leaf of Sugarcane cv R579 were infected with Agrobacterium::pKYS, and were grown on MS agar media to regenerate transgenic plants. Regenerated plants were planted on soil and then analyzed. The transformed E. coli could express SoSPS1 gene, that in this experiment produced an active SoSPS1 protein. Based on PCR analysis, three lines of transformant tobacco were obtained (line 3, 13, 17), that positively harboring transgene of SoSPS1. The SPS activites of tobacco line 3, 13 and 17 increased up to 114.965; 165.765 and 118.49%, respectively. The sucrose content in leaf of line 3 and 13 increased up to 118,49% and 174,69%, whereas in line 17 decreased up to 93,64%. Based on PCR analysis, four lines of transformant sugarcane (line 4, 5, 6, 7) were obtained that containing transgene SoSPS1. Western blot analysis indicated that SPS transgene could be expressed at translation level and produced higher amount of SPS protein than control sugarcane. The SPS activities in leaf of sugarcane of line 4, 5, 6 and 7 were higher than the control, that increased up to 140,85%; 228,17%; 173,94% and 280,28%, respectively. The sucrose content in leaf increased up to 216,28%; 193,02%; 188,37% and 176,74% respectively, and followed by the increased ratio between sucrose/starch content. The increasing SPS activity in the transformants sugarcane leaf were followed by increased the activity of sucrose hydrolyzing enzyme, primarily SS and AI. The sugarcane transformants that express SoSPS1 gene had higher stalk sucrose content than the control sugarcane. In sugarcane transformant first generation, K1 and B1 generation of sugarcane transformants, too high SPS activity caused reducing sucrose content in leaf.

Kata Kunci : Biosintesis Sukrosa,Tanaman Tebu,Transfer Gen SPS, sucrose, over-expression, SPS, Agrobacterium, sugarcane, tobacco, E. coli