Isolation, cloning, and expression analysis of gene coding copper/zinc superoxide dismutase (CuZn-SOD) from Melastoma malabathricum L.

Abstract

Melastoma malabathricum L. is an indigenous plant in tropical Southeast Asia with exceeding tolerance to aluminum (Al) stress in acid soils. Although increasing evidence suggests that Al-stress is accompanied by oxidative damages in plants, little has been described on the antioxidative components in this tolerant plant. Copper/zinc SOD (CuZn-SOD) is one of aluminum-induced genes. Analysis of the expression of genes induced by Al in M. malabathricum requires internal control genes. Actin is belong to housekeeping genes commonly used as an internal control. This study aimed to isolate and clone the cDNA fragment of MmACT encoding for actin of M. malabathricum, and isolate, clone and expression analysis of copper/zinc SOD (CuZn-SOD) from M. malabathricum L. Total RNA was isolated and used as template for cDNA synthesis by reverse transcription. Four of cDNAs clones were isolated, and were called MmACT1, MmACT2, MmACT3, and MmACT4. The size of MmACT1 and MmACT2 is 617 bp, whereas MmACT3 and MmACT4 is 735 bp. The sequence of MmACT cDNAs was registered in GenBank / EMBL / DDBJ database with accession numbers AB500686, AB500687, AB500688, and AB500689. Full length cDNA of MmCuZn-SOD had been successfully isolate by RACE. The size of MmCuZn-SOD is 824 bp that encoded a 152-amino acid polypeptide with a predicted pI value of 5.77, and showed high homology to other cytosolic CuZn-SODs from higher plants. Semi-quantitative RT-PCR showed that MmCuZn-SOD mRNA was highly expressed in the leaf tissues than stem and root. The Al treatment strongly induced the expression of MmCuZn-SOD both in the leaf and root tissues. These results suggested the fortification of the anti-oxidative defense system under Al stress in this acid soil-tolerant wild plant. Overexpression vectors was successfully constructed and each has been introduced into Agrobacterium tumefaciens LBA 4044 by triparental mating (TPM). N.benthamiana and Nicotiana tabacum transgenics had been obtained by mediated-A. tumefaciens. Analysis of segregation of T1 transgenic plants based on analysis of Khi-Quadrat (2) showed that the T1 transgenic plants accordance with 3:1 of Mendelian inheritance pattern. RNAi vector had been successfully constructed using GATEWAYTM cloning technology and it had been introduced into M. malabathricum L. mediated by Agrobacterium tumefaciens LBA 4404. Transgenic plants analyzes to Al stress showed that in the MS medium containing 1 mM Al (AlCl36H2O), the transgenic plants underwent growth suppression, whereas non-transgenic plants underwent growth normally. These results suggested that suppression of MmCuZn-SOD gene expression by RNAi to M. malabathricum L. caused the plant became sensitive to Al

Melastoma malabathricum L. merupakan tanaman asli Asia Tenggara yang toleran terhadap cekaman aluminium (Al) pada tanah asam. Gen-gen yang ekspresinya diinduksi oleh Al diduga terlibat dalam sistem toleransi terhadap Al. Gen superoxside dismutase (SOD) merupakan salah satu gen yang ekspresinya diinduksi Al. SOD diduga memiliki peran dalam ketahanan M. malabathricum. Untuk mempelajari ekspresi gen secara kuantitatif pada M. malabathricum L, informasi housekeeping gene dari tumbuhan tersebut juga sangat dibutuhkan sebagai kontrol internal. Salah satu housekeeping gene yang populer dijadikan sebagai internal kontrol adalah gen aktin. Penelitian ini bertujuan mengisolasi dan mengklon fragmen gen penyandi aktin dari M. malabathricum L, selanjutnya melakukan isolasi, pengklonan, dan analisis ekspresi gen penyandi copper/zinc superoxide dismutase (CuZn-SOD) dari M. malabathricum L, dan mempelajari ekspresi gen MmCuZn-SOD melalui konstruksi ekspresi berlebih (overekspresi) di tanaman model Nicotiana benthamiana dan Nicotiana tabacum, dan pembungkaman gen MmCuZn-SOD melalui RNAi di M. malabathricum L. RNA total telah berhasil diisolasi dan dijadikan sebagai cetakan untuk sintesis cDNA total melalui transkripsi balik. Empat fragment cDNA yang menyandi aktin dari M. malabathricum telah berhasil diisolasi dan disisipkan ke dalam plasmid pGEM-T Easy. Keempat fragmen ini selanjutnya dinamakan fragmen MmACT1, MmACT2, MmACT3, dan MmACT4. Analisis urutan nukleotida menunjukkan bahwa fragmen MmACT1 dan MmACT2 berukuran 617 pb, dan fragmen MmACT3 dan MmACT4 berukuran 735 pb. Antar keempat fragmen cDNA MmACT ini memiliki kemiripan nukleotida sekitar 78%-99%, dan kemiripan asam amino sekitar 98% - 100%. Analisis hubungan filogenetik berdasarkan urutan asam amino menunjukkan bahwa pada ketidakmiripan 1% MmACT1, MmACT2, MmACT3 mengelompok dengan ACT5 Populus trichocarpha, sementara MmACT4 mengelompok dengan ACT9 P. trichocarpa dan ACT1 Gossypium hirsutum, dan kedua kelompok ini terpisah dengan aktin dari tumbuhan monokotil. Keempat fragmen MmACT ini telah didaftarkan di bank data GenBank/EMBL/DDBJ dengan nomor aksesi AB500686, AB500687, AB500688, and AB500689. Selanjutnya fragmen cDNA yang menyandi CuZn-SOD dari M. malabathricum juga telah berhasil diisolasi. Gen utuh MmCuZn-SOD yang berhasil diisolasi dengan metode RACE berukuran 824 pb, terdiri dari 459 pb open reading frame (ORF) yang menyandi 152 asam amino dengan prediksi nilai pI 5.77. Analisis filogenetik berdasarkan urutan asam amino menunjukkan bahwa MmCuZn-SOD memiliki kemiripan yang tinggi dengan CuZn-SOD sitosol tanaman tingkat tinggi. Berdasarkan analisis semi-kuantitatif RT-PCR menunjukkan bahwa MmCuZn-SOD terekspresi pada jaringan daun, batang dan akar. Perlakuan Al menginduksi ekspresi MmCuZn-SOD baik dalam jaringan daun maupun akar