Isolasi dan Identifikasi Bakteri Penghasil ACC Deaminase Asal Rizosfer Nanas Cekaman serta Deteksi, Ekspresi, dan Kelimpahan Gen acdS-nya
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Date
2018Author
Jaya, Dori Kusuma
Giyanto
Nurhidayat, Novik
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ACC deaminase merupakan enzim sitoplasma yang berperan penting dalam menekan kadar etilen berlebih tanaman yang dilanda cekaman dengan cara menghidrolisis senyawa pembentuknya, ACC. Penelitian ini bertujuan untuk mendapatkan isolat dan identitas bakteri penghasil ACC deaminase, primer gen acdS, tingkat ekspresi gen acdS, dan kelimpahan gen acdS pada tanah rizosfer nanas yang dilanda cekaman. Empat jenis sampel tanah asal bakteri diperoleh meliputi tanah dengan cekaman herbisida, cekaman Phytopthora spp., cekaman genangan, dan tanah noncekaman.
Penelitian ini diawali dengan mengisolasi bakteri rizosfer pada media umum TSA (Tryptic Soy Agar) kemudian ditumbuhkan kembali pada media selektif DF (Dworkin-Foster) mengandung senyawa ACC. Bakteri yang tumbuh pada media selektif ini diindikasikan memiliki enzim ACC deaminase yang disandi oleh gen acdS. Tahap selanjutnya adalah melakukan identifikasi molekuler isolat bakteri tersebut berdasarkan gen 16S rRNA. Selanjutnya, tahap ekstraksi DNA dan RNA isolat bakteri serta DNA bakteri total tanah dilakukan sebagai template pada sistem deteksi berbasis real time PCR.
Sebanyak 49 isolat bakteri berhasil tumbuh pada media TSA dan hanya 7 isolat yang mampu tumbuh pada media selektif DF mengandung 3 mM L-1ACC. Hasil identifikasi gen 16S rRNA menunjukkan bahwa keempat isolat 3, 2A, 5B, dan 1E dengan pertumbuhan terbaik termasuk ke dalam Stenotrophomonas maltophilia, Burkholderia territorii, Pseudomonas oryzihabitans, dan Bacillus tropicus. Sepasang primer 105F-acdS 5’-TGCCAAGCGTGAAGACTGC-3’ and 244R-acdS 5’-GGGTCTGGTTCGACTGGAT-3’ berhasil mengamplifikasi sekuens parsial acdS sepanjang 140 pb pada isolat 3, 2A, 5B, dan 1E dengan suhu disosiasi berturut-turut 86, 89, 87, dan 89.5oC berdasarkan analisis kurva melt peak dan dikonfirmasi dengan pelacakan BlastN dan BlastX. Berdasarkan analisis filogenetik, sekuens acdS isolat 2A dan 1E memiliki kemiripan yang lebih tinggi dibandingkan dengan isolat 3 dan 5B. Sedangkan tingkat ekspresi gen acdS pada keempat bakteri berturut-turut adalah B. tropicus (17.24), B. territorii (17.86), P. oryzihabitans (20.87), dan S. maltophilia yang tidak menunjukkan nilai Cq. Analisis DNA sampel tanah menyimpulkan bahwa kelimpahan gen acdS tanah noncekaman berbeda signifikan terhadap tanah cekaman herbisida dan Phytopthora spp. kecuali pada tanah cekaman genangan. Kelimpahan gen acdS antarcekaman secara umum berbeda signifikan kecuali pada sampel CPB terhadap CGB. Sedangkan kelimpahan gen acdS antara sampel tanah tanaman sehat dan sakit tidak berbeda signifikan kecuali pada sampel cekaman Phytopthora spp. Hasil penelitian ini menggambarkan bahwa bakteri rizosfer asal rizosfer nanas memliki gen acdS yang menyandi ACC deaminase dan diduga mampu meregulasi kadar etilen tanaman dalam merespon berbagai cekaman lingkungan. ACC deaminase is a cytoplasmic enzyme which play an important role in lowering overproduced ethylene in stressed plant by cleaving its precursor, ACC. The aim of this study was to obtain ACC deaminase-encoding rhizobacterial isolates and identity, acdS gene primers, acdS gene expression among bacterial isolates, and acdS gene abundance in stress soil of pineapple rhizosphere. Four types of soil samples where bacterial isolates obtained include herbicide-stress soil, flooding-stress soil, Phytopthora spp.-stress soil and nonstress soil.
This study was initiated by isolating rhizobacteria on the TSA (Tryptic Soy Agar) medium and then re-growing on DF (Dworkin-Foster) selective medium containing 3 mM L-1ACC compound. Bacteria that grow on this selective medium were indicated to have ACC deaminase enzyme encoded by acdS gene. Following the purified isolates, the selected isolates were then molecularly identified based on 16S rRNA gene. The DNA and RNA from bacterial isolates and total soil bacterial DNA were extracted and then used as a template on real time PCR reaction.
A total of 49 bacterial isolates were successfully grown on TSA medium and only 7 isolates were able to grow on DF selective medium containing 3 mM L-1 ACC. Based on 16S rRNA gene, the four best growth isolates marked 3, 2A, 5B, and 1E belong to Stenotrophomonas maltophilia, Burkholderia territorii, Pseudomonas oryzihabitans, dan Bacillus tropicus, respectively. A pair of primers 105F-acdS 5'-TGCCAAGCGTGAAGACTGC-3'and 244R-acdS 5'-GGGTCTG-GTTCGACTGGAT-3' successfully amplified the 140 bp of partial acdS sequence in isolates 3, 2A, 5B, and 1E with dissociation temperature at 86, 89, 87, and 89.5oC respectively based on melt peak analysis and then confirmed by sequencing along with BlastN and BlastX search. Based on phylogenetic analysis, the acdS sequences of isolates 2A and 1E had a higher similarity compared to that of isolates 3 and 5B. While the acdS gene expression level in the four bacteria based on Cq values from higher to lower are Bacillus tropicus (17.24), Burkholderia territorii (17.86), Pseudomonas oryzihabitans (20.87), and Stenotrophomonas maltophilia which did not show Cq value. DNA analysis of soil samples conclude that the abundance of the acdS gene in nonstress soil had a significant difference compared to that of herbicide-stress soil and Phytopthora-stress soil except flooding-stress soil. The abundance acdS genes interstress-soil samples showed a significant difference except in sample CPB to CGB . While the abundance of the acdS gene between good and bad plant did not show significant differences except in Phytopthora spp.-stressed soil. The results of this study illustrate that rhizobacteria obtained from pineapple rhizosphere possess the acdS gene that encodes ACC deaminase and are estimated to be able to regulate the plant ethylene level in response to various environmental stresses.
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