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All Journal JURNAL PEMULIAAN TANAMAN HUTAN Indonesian Journal of Biotechnology Journal of Tropical Biodiversity and Biotechnology Agrin : Jurnal Penelitian Pertanian Journal of Tropical Crop Science Menara Perkebunan Nusantara Science and Technology Proceedings

Riza Arief Putranto

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Author-ID : 3618383

Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Economics, Econometrics & Finance Engineering Environmental Science Immunology & microbiology Law, Crime, Criminology & Criminal Justice Materials Science & Nanotechnology Medicine & Pharmacology

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Characterization of a Drought-Inducible Dehydrin Promoter from Sugarcane (Saccharum officinarum L.) in Tobacco (Nicotiana tabacum L.) Iskandar, Hayati Minarsih; Suhandono, Sonny; Pambudi, Jembar; Kristianti, Tati; Putranto, Riza Arief; Mose, Windi; Sustiprijatno, Sustiprijatno
Journal of Tropical Crop Science Vol 7 No 01 (2020): Journal of Tropical Crop Science
Publisher : Department of Agronomy and Horticulture, IPB University

Dehydrin (DHN) is known to play an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). Previous research reported the increased expression of DHN in sugarcane stems exposed to drought stress for 15 days which may be controlled by its corresponding stress inducible promoter. The DHN promoter was succesfully isolated from sugarcane variety PSJT 941 (Pr-1DHNSo) and was cloned to pBI121 expression vector fused to a β-glucuronidase (GUS) reporter gene. The aim of this research was the functional testing of the Pr-1DHNSo promoter through transformation into tobacco plant treated with in vitro drought stress. Genetic transformation of Pr-1DHNSo construct was conducted by Agrobacterium tumefaciens. The transformed tobacco was then subjected to drought stress treatment using 40% PEG 6000 for five sequential incubations (0, 12, 24, 48 and 72 hours). The GUS assay reveal that the transformed tobacco treated with drought stress showed a blue color denoting GUS activity in leaf, stem and root tissues and this expression increased along with the length of the drought treatment. The analysis of gusA gene using real time-qPCR normalized to the L25 reference gene also showed that the expression increased in line with the length of time of drought stress. The results presented in this study indicated that the Pr-1DHNSo promoter from sugarcane was expressed and induced by drought stress treatment in tobacco.

IDENTIFIKASI TIGA ISOLAT CENDAWAN PENGHASIL GAHARU DARI NUSA TENGGARA BARAT DENGAN MENGGUNAKAN PRIMER ITS DAN TEF 1-α Y.M.M Anita Nugraheni; Lutfi Anggadhania; Riza Arief Putranto
Jurnal Pemuliaan Tanaman Hutan Vol 9, No 2 (2015): Jurnal pemuliaan Tanaman Hutan
Publisher : Center for Forest Biotechnology and Tree Improvement (CFBTI)

Several isolates of agarwood-forming fungus in Gyrinops versteegii have been isolated from the result of exploration in Lombok and Sumbawa Islands. This study aimed to identify the three fungus associated with the agarwood formation in G. versteegii originated from Lombok Tengah, Alas, and Lombok Barat, Nusa Tenggara Barat. The three fungus cultured in liquid medium PDB (Potato Dextrose Broth) and incubated for 1 month in shaken culture. The mycelium of each fungus was harvested for DNA isolation. Amplification is done by Polymerase Chain Reaction (PCR) using the Primers ITS and TEF with obtained amplicon having base length ranging of 300-600 bs. BLAST analysis showed that the three fungus have similarity with Fusarium solani. These results were confirmed by phylogenetic tree where all fungus has genetic relationship with F. solani.

CRISPR/Cas9‐mediated knockout of an oil palm defense‐related gene to the pathogenic fungus Ganoderma boninense Asmini Budiani; Imam Bagus Nugroho; Dini Astika Sari; Inez Palupi; Riza Arief Putranto
Indonesian Journal of Biotechnology Vol 24, No 2 (2019)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/ijbiotech.52170

Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense. Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense. A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense.

Respons molekuler Hevea brasiliensis ethylene response factors (HbERFs) sebagai marka ekspresi gen terhadap stimulasi ethephon pada klon-klon tanaman karet Moleculer response of Hevea brasiliensis ethylene response factors (HbERFs) as expression marker genes in response to ethephon stimulation in rubber tree clones Riza Arief PUTRANTO; . KUSWANHADI; Pascal MONTORO
E-Journal Menara Perkebunan Vol 82, No 2: Desember 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Abstract Real-Time quantitative RT-PCR technique is a sensitive method for measuring the accumulation of gene transcripts. This widely used technique in a variety of plant species; including rubber tree (Hevea brasiliensis) must meet basic criteria in order to produce accurate gene expression markers. Gene expression markers associated to the response of ethephon stimulation such as the Hevea brasiliensis Ethylene Response Factors (HbERFs) family has been characterized in a single rubber clone. It is known that the effect of genotype on rubber tree clones can give different expression of the same gene. This difference can be converted into a profile that characterizes clones to a certain trait. This study aimed to identify gene expression profile in response to ethephon stimulation using six HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1) in three rubber tree clones having contrasted latex metabolism (PB 260, SP 217, and RRIM 600). Total RNA was isolated from 18 samples and used for cDNA synthesis. The quality of cDNAs was examined by PCR using HbActin primer. HbRH2b was selected among the 11 housekeeping genes to be used as an internal control in gene expression analysis. Gene expression analysis resulted to an induction and inhibition of HbERFs by ethephon stimulation which are specific to a particular clone. Expression profile of three Hevea clones showed distinct characteristics. The high latex metabolism clone PB 260 was characterized by the upregulated expression of HbRAP2.3 and HbERF12. The low latex metabolism clone SP 217 was characterized by the upregulated expression of HbRAP2.3 and HbRRTF1. Meanwhile, the profile of intermediate latex metabolism clone RRIM 600 was shown by downregulated expression of HbORA47 and up-regulated expression of HbABR1. This study shows that HbERFs gene family is an important expression marker because it can inform physiological conditions of rubber clones associated in response to ethephon. AbstrakTeknik Real-Time quantitative RT-PCR merupakan metode sensitif untuk mengukur akumulasi transkrip dari gen. Teknik yang telah banyak digunakan pada berbagai spesies tanaman, termasuk tanaman karet (Hevea brasiliensis) ini harus memenuhi kriteria dasar agar meng-hasilkan marka ekspresi gen yang akurat. Beberapa marka ekspresi gen terkait respons terhadap stimulasi ethephon seperti famili gen Hevea brasiliensis Ethylene Response Factors (HbERFs) telah dikarakterisasi pada satu klon tanaman karet. Sebagaimana diketahui, efek genotip pada klon tanaman karet dapat memberikan ekspresi yang berbeda dari gen yang sama. Perbedaan ekspresi tersebut dapat dikonversi menjadi sebuah profil yang menjadi karakteristik klon karet terhadap perlakuan tertentu. Penelitian ini bertujuan untuk mengidentifikasi profil ekspresi gen HbERFs pada tiga klon tanaman karet (PB 260, SP 217, dan RRIM 600) yang memiliki metabolisme lateks yang berbeda terhadap respons stimulasi ethephon dengan menggunakan enam gen HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1). RNA total diisolasi dari 18 sampel dan digunakan untuk sintesis cDNA. Kualitas cDNA diperiksa dengan PCR menggunakan primer HbActin. Gen HbRH2b terseleksi diantara 11 gen housekeeping digunakan sebagai kontrol internal pada analisis ekspresi gen. Hasil dari analisis ekspresi gen menunjukkan bahwa stimulasi ethephon memiliki efek induksi dan inhibisi gen yang spesifik untuk klon tertentu. Profil ekspresi dari tiga klon tanaman karet yang diuji memperlihatkan perbedaan karakteristik. Klon metabolisme tinggi PB 260 ditunjukkan dengan ekspresi positif dari gen HbRAP2.3 dan HbERF12. Klon meta-bolisme rendah SP 217 ditunjukkan oleh ekspresi positif gen HbRAP2.3 dan HbRRTF1. Sedangkan klon metabo-lisme intermedier RRIM 600 memiliki profil ekspresi negatif dari HbORA47 dan ekspresi positif dari HbABR1. Penelitian ini memperlihatkan bahwa famili gen HbERFs merupakan marka ekspresi yang penting karena dapat menginformasikan kondisi fisiologis klon tanaman karet terkait respons terhadap ethephon.

Kloning dan karakterisasi daerah promoter gen penyandi ADP glucose pyrophosphorylase dari Metroxylon sagu rendemen pati-tinggi dan -rendah [Cloning and characterization of promoter region of ADP glucose pyrophosphorylase-encoding gene from Metroxylon sagu with high- and low-starch content] Asmini BUDIANI; Riza Arief PUTRANTO; Hayati MINARSIH; Imron RIYADI; . SUMARYONO; Barahima ABBAS
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v84i1.200

ADP-glucose pyrophosphorylase (AGPase) is one of the key enzymes in the starch biosynthesis. In many plants, the activity of this enzyme was reported to affect the yield and composition of the produced starch. This research is a part of an effort to develop molecular markers for early selection of high starch-yielding of sago palm. The purpose of the research was to isolate promoters of AGP gene and to analyze the differences in their DNA sequences between sago palm with high starch content (MsHS) and low starch content (MsLS). DNA was isolated and purified from the leaves of the two sago palm. The promoter region of AGP was amplified by Genome Walking technique. The specific primers were designed by Primer3 program based on the information of DNA sequence of AGP genes of sago palm from previous studies. Selected DNA fragments resulted from Genome Walking were isolated from the gel, cloned into E. coli, and analyzed its DNA sequence. DNA sequence analysis showed that one DNA fragment from MsHS (± 1500 bp) and one DNA fragment from MsLS (> 2000 bp) were confirmed as a 5’ upstream of the AGP gene. Further in silico analysis using MEME program identified various DNA motifs of cis-acting elements, which confirmed that those DNA fragment were promoter region of the gene. Preliminary analysis showed the differences in DNA sequences and motives of cis-acting elements in the promoter region of the two samples which might influence or indirectly associated with the character of the starch yield in sago palm.

The Hevea brasiliensis AP2/ERF superfamily: from ethylene signalling to latex harvesting and physiological disease response Riza Arief PUTRANTO; Pascal MONTORO
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Ethylene is a hormone known for its involvement in the process of latex harvesting in Hevea brasiliensis. It facilitates latex flow by activation of endogenous metabolism in the anastomosed latex cells called laticifers. In regard to its ambivalent role, ethylene is both favourable to the latex production and unfavourable, to a certain level, to the apparition of a physiological disease termed as tapping panel dryness (TPD). Comprehensive researches have been carried out to reveal the molecular actors in ethylene biosynthesis and signalling pathways in Hevea brasiliensis. One of the most important superfamily implicated as the last transcription factor known in plant ethylene signalling is the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF). Currently, 114 unique sequences related to the Hevea AP2/ERF gene superfamily have been identified and characterized. Specific characterizations under the condition of harvesting stress and the occurrence of TPD have identified 36 gene expression markers (GEMs). Eighteen of these GEMs were predicted as ortholog with 19 Arabidopsis AP2/ERF genes. The characterization was mainly focused on transcriptional regulation, whilst potential post-transcriptional and post-translational regulations of HbAP2/ERF genes were formerly predicted. Three HbERF groups (HbERF-VII, HbERF-VIII and HbERF-IX) were hypothesized to have an important role in Hevea tolerance during latex production as they highly accumulated in laticifers and in response to multiple abiotic stresses. Further functional analysis of several key genes is suggested in order to fully understand the regulation of HbAP2/ERFs. Finally, the molecular markers for future Hevea breeding could be possibly developed from this superfamily.

Evaluasi 18 primer SSR untuk pengembangan sidikjari DNA tanaman karet (Hevea brasiliensis Muell. Arg.) Evaluation of 18 SSR primers to develop DNA fingerprint of rubber tree (Hevea brasiliensis Muell. Arg.) Asmini BUDIANI; Sekar WOELAN; Hayati MINARSIH; . NUHAIMI-HARIS; Riza Arief PUTRANTO
E-Journal Menara Perkebunan Vol 82, No 2: Desember 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Abstract Breeding program of rubber tree to produce elite clones is hampered by the length of selection cycles. On the other hand, attempts to increase production by extensification of the plantation area is also facing a problem from the availability of the rootstock, causing the occurence of fake clones without any information of their origin. Therefore, the availability of molecular markers to be used as DNA fingerprint of rubber tree clones is needed. This will help the breeder to shorten the length of selection program and to identify the purity of the clone. This research was aimed to evaluate 18 SSR primer pairs that had been published to identify 17 rubber clones. Pure genomic DNAs were isolated from 17 clones, followed by experiment to optimize annealing tempe-rature for each primer to obtain the best amplification product. Initially, the PCR product was run in both the agarose and polyacrylamide gels. However, the analysis of all PCR products were then conducted on SDS polyacrylamide gel, since this gel can separate DNA fragments with only a few bases differences. The results showed that 14 clones have been identified specifically using 11 primers. Four out of 18 primer pairs used could identify 12 rubber tree clones, which are PR 107, PR 261, SP 217, PB 330, PB 340, IRR 5, IRR 112, IRR 118, IRR 220, GT 1, BPM 101 and RRIM 712. Each clone can be distinguished from each other using only one primer pair. Identification of the other tree clones (PB 5/51, PB 260, and RRIC 110) has to be conducted by combining the several PCR products using different primer pairs. Although these results showed that SSR markers had high potential to be used as DNA fingerprint on rubber tree clones, the set of the primer pairs should be tested among other clones, as well as other SSR primers should be tested to identify the clones which could not be identified using the 18 primer pairs in this experiment.Abstrak Pemuliaan tanaman karet untuk menghasilkan klon-klon unggul baru menghadapi masalah lamanya siklus seleksi. Di sisi lain, upaya peningkatan produksi melalui pembukaan lahan baru, juga terkendala oleh ketersediaan bibit, yang memicu beredarnya bibit palsu, yang umumnya tidak jelas asal usulnya. Oleh karena itu, diperlukan ketersediaan marka yang dapat digunakan sebagai sidikjari DNA bagi klon-klon tanaman karet yang ada, sehingga dapat membantu mempercepat proses seleksi dan mengetahui kemurnian bibit. Penelitian ini bertujuan untuk mengevaluasi 18 primer SSR yang telah dipublikasikan untuk mengidentifikasi 17 klon karet. DNA yang murni diisolasi dari 17 klon, kemudian dilakukan optimasi suhu annealing untuk setiap jenis primer agar diperoleh hasil amplifikasi terbaik. Pada awal percobaan hasil PCR dicek pada gel agarosa dan gel poliakrilamida, namun analisis untuk seluruh hasil PCR dilakukan pada gel SDS poliakrilamid, karena gel ini secara nyata dapat memisahkan fragmen DNA yang hanya berbeda beberapa basa. Hasil percobaan menunjukkan bahwa 14 klon dapat diidentifikasi secara spesifik menggunakan 11 primer. Empat dari 18 pasang primer yang diuji dapat mengidentifikasi 12 klon yang dianalisis, yaitu PR 107, PR 261, SP 217, PB 330, PB 340,. IRR 5, IRR 112, IRR 118, IRR 220, GT 1, BPM 101 dan RRIM 712. Masing-maing klon tersebut dapat dibedakan dari klon lainnya hanya dengan menggunakan satu jenis primer. Sedangkan identifikasi tiga klon lainnya (PB 5/51, PB 260, dan RRIC110) harus dilakukan dengan menggabungkan hasil PCR menggunakan beberapa primer. Meskipun hasil percobaan ini menunjukkan bahwa marka SSR sangat berpotensi untuk digunakan sebagai sidikjari DNA klon-klon karet, namun primer yang sama perlu diuji untuk klon-klon lainnya. Demikian pula primer lain perlu diuji untuk mengidentifikasi klon-klon yang belum teridentifikasi menggunakan 18 primer dalam penelitian ini.

The in silico study of the COBRA gene family in sugarcane related to potential biomass content Riza Arief PUTRANTO; Galuh Wening PERMATASARI; Rizka Tamania SAPTARI
E-Journal Menara Perkebunan Vol 90, No 1 (2022): April, 2022
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v90i1.486

AbstractSugarcane (Saccharum sp.) is potential as a biofuel and biomaterial source for its high cellulose content. Cellulose is the main constituent of the plant cell wall, as a linear chain arranged in a polysaccharide bundle, called cellulose microfibril. A gene named COBRA has been revealed to play role in the orientation of microfibril and cellulose crystallization. The COBRA gene in the Saccharum spp is under-explored. Therefore, the in silico study was conducted to explore the COBRA gene in Saccharum sp. By comparative genomics methods, the COBRA genes from Arabidopsis sp. (AtCOBLs) were compared to the Saccharum sp. (SoCOBLs). The conserved domain was then identified and the cluster system was constructed under a phylogenetic tree. Furthermore, each SoCOBLs protein was modelled to analyze its structure. According to the analysis, eleven of Saccharum sp. genomic scaffolds were successfully identified. Moreover, conserved domain identification resulted in nine SoCOBLs proteins. The phylogenetic tree showed two main clusters: I and II, differentiating those COBLs families based on the protein sequence, domain motif and amino acid properties. It leads to the variation of SoCOBLs protein structure as the results of the amino acid properties. Overall, the COBRA gene has been identified genomically in Saccharum sp. Yet, the function and tissue-specific expression are still unclear. It was predicted to act as the regulator of microfibril orientation and the cellulose synthesis process. Hence, further analyses by in vitro and in vivo are indispensable.[Keywords: cellulose, comparative genomic, Saccharum sp.]AbstrakTanaman tebu (Saccharum sp.) berpotensi sebagai sumber bahan bakar nabati dan biomaterial karena kandungan selulosanya yang tinggi. Selulosa merupakan komponen utama penyusun dinding sel tanaman, sebagai rantai lurus yang tersusun dalam gugusan polisakarida, yang disebut mikrofibril selulosa. Sebuah gen bernama COBRA telah diketahui berperan dalam menentukan arah mikrofibril dan kristalisasi selulosa. Gen COBRA pada spesies Saccharum spp. belum banyak dipelajari. Oleh karena itu, kajian in silico dilakukan untuk mempelajari gen COBRA pada Saccharum sp. Melalui metode perbandingan genomika, gen COBRA dari Arabidopsis sp. (AtCOBLs) dibandingkan dengan gen COBRA dari Saccharum sp. (SoCOBLs). Domain conserve pada gen kemudian diidentifikasi dan sistem klaster disusun dalam sebuah pohon filogeni. Setelah itu, dibuat model untuk menganalisis struktur dari protein SoCOBL. Dari hasil analisis, sebelas perancah genom Saccharum sp. berhasil diidentifikasi. Kemudian, identifikasi daerah lestari menghasilkan sembilan protein SoCOBL. Pohon filogeni menggambarkan dua klaster utama: I dan II, yang membedakan famili SoCOBLs tersebut berdasarkan sekuens protein, motif domain, dan karakteristik asam amino. Karakteristik asam amino menyebabkan variasi pada struktur protein-protein SoCOBL. Secara umum, gen COBRA telah teridentifikasi pada Saccharum sp., meskipun fungsi dan ekspresi spesifiknya pada jaringan masih belum diketahui. Diperkirakan gen tersebut berperan sebagai pengatur arah mikrofibril dan proses sintesis selulosa. Oleh karena itu, perlu adanya analisis lebih lanjut pada level in vitro dan in vivo.[Kata kunci: selulosa, genomika komparatif, Saccharum sp.]

Analisis ko-ekspresi gen-gen regulasi upstream dari gen Dehydrin di tanaman tebu (Saccharum officinarum L.) pada kondisi cekaman kekeringan Hayati MINARSIH; Jembar PAMBUDI; Riza Arief PUTRANTO
E-Journal Menara Perkebunan Vol 88, No 2 (2020): Oktober,2020
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v88i2.396

Sugarcane plantations in Indonesia have been expanded and shifted to the marginal land characterized by long drought period, therefore, an attempt has been initiated to generate drought tolerance varieties through genetic engineering. It could be conducted by inserting the gene that involve in plant adaptation response to drought stress such as dehydrin (DHN) into sugarcane genome. The promoter of sugarcane DHN gene was isolated and transformed into sugarcane in the previous research. This study aimed to demonstrate the functionality of sugarcane DHN promoter through expression analysis of DHN regulatory genes that play a role in response to drought stress. Expression analyses using RT-qPCR were also conducted on regulatory genes of sugarcane that inserted by Pr-1DHNSo construct treated with drought stress. The results showed that the expressions of SoMYB, SoWRKY, SoNAC, and SoDHN genes were escalated on sugarcane 16 days after stress treatment ranging from 353 to 4067 folds relatively to untreated samples in which SoNAC gene showed the highest expression. On the other hand, the analysis on transgenic sugarcane carrying DHNpromoter construct showed SoNAC and SoDREB expression increased after 72 hours under drought stress. The expression values of SoNAC in transgenic and non-transgenic plants under drought condition were 4.79 and 4.99, respectively. Meanwhile, the expression values of SoDREB in transgenic and non-transgenic plants under drought condition were 13.2 and 13.3, respectively. The results of these experiments showed that the promoter construct of Pr-1DHNSo was induced by drought stress treatments highlighting the regulation of several upstream genes of SoDHN.

Ekspresi dan kloning gen penyandi ADP-Glucose Phyrophosphorylase dari tanaman sagu (Metroxylon sagu Rottb.) Expression and cloning of gene encoding ADP-Glucose Phyrophosphorylase from sago palm (Metroxylon sagu Rottb.) Asmini BUDIANI1; Riza Arief PUTRANTO; Hayati MINARSIH; Imron RIYADI; . SUMARYONO; Barahima ABBAS
E-Journal Menara Perkebunan Vol 83, No 2: Desember 2015
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

AbstractSago palm (Metroxylon sagu Rottb.) is a potential food and energy resources becouse it is the highest starch producing plant. Breeding of sago palm should be directed to produce elite genotype with superior characters such as high starch content, wider pith diameter, without spine and high starch quality. However, research on sago palm in Indonesia so far is limited espescially in the field of cultivation and breeding, and attempt to produce such elite would take long time. Availability of molecular marker for starch content would be beneficial to shorten the length period of breeding. ADP-Glucose Phyrophosphorylase is one of the important enzymes in starch biosynthesis. Therefore its gene is an interesting subject in order to develope molecular marker of high starch content. This research was aimed to study the expression of gene encoding AGP in the sago palm with high starch content versus low starch content, and to clone the full cds of the gene. RNA was isolated from leaf and pith of both palms. Exspression analysis and amplify-cation of full cds were conducted by Reverse Transcryptase-Polymerase Chain Reaction (RT-PCR) using specific primers. The results showed that sago palm with higher starch content expressed AGP higher than that of sago palm with lower starch content. Expression of AGP in the full developing leaf was higher than in the young leaf, and there was no expression detected in the pith. The full cds of AGP was successfully amplified and cloned. Even though the DNA sequence showed high homology with DNA sequence of the same gene that has been deposited in GenBank, there were differences in severall nucleotide including that in the active domain of the enzyme.AbstrakTanaman sagu merupakan sumber pangan dan energi yang sangat potensial untuk dikembangkan karena merupakan tanaman penghasil karbihidrat tertinggi. Pemuliaan tanaman sagu mestinya diarah-kan untuk menghasilkan bibit sagu yang selain memiliki rendemen pati tinggi, juga memiliki diameter empulur besar, tidak berduri dan memiliki cita rasa pati yang enak. Namun, sampai saat ini riset mengenai sagu di Indonesia masih sangat terbatas, sehingga pemuliaan sagu untuk menghasilkan bibit unggul demikian akan memerlukan waktu lama. Ketersediaan penanda rendemen pati akan sangat membantu mempercepat pemuliaan tanaman tersebut. ADP-Glucose Pyrophosphorylase adalah salah satu enzim yang berperan penting dalam biosintesis pati, sehingga gene penyandinya merupakan subjek yang menarik dalam pengembangan marka kandungan pati tinggi. Sebagai bagian dari upaya untuk mendapat-kan penanda rendemen pati tinggi pada tanaman sagu, penelitian ini bertujuan untuk mempelajari ekspresi gen penyandi AGP. RNA diisolasi dari daun tanaman sagu rendemen pati rendah dan tanaman sagu rendemen pati tinggi. Perbedaan tingkat ekspresi gen penyandi AGP dari tanaman sagu rendemen pati tinggi vs rendemen pati rendah, dianalisis dengan teknik Reverse-Transcryptase PCR menggunakan primer spesifik. Hasil penelitian menunjukkan bahwa tanaman sagu rendemen pati tinggi mengekspresikan AGP lebih tinggi dibandingkan dengan tanaman sagu rendemen pati rendah. Ekspresi gen tersebut pada daun tua (full developing leaf) lebih tinggi di-bandingkan dengan pada daun muda, dan pada empulur tidak dideteksi ekspresi gen tersebut. Daerah penyandi lengkap AGP subunit kecil telah diklon. Meskipun memiliki homologi yang tinggi dengan sekuen DNA gen yang sama yang telah dideposit pada GenBank, namun terdapat perbedaan beberapa nukleotida termasuk pada daerah domain aktif dari enzim tersebut.

Co-Authors . KUSWANHADI . NUHAIMI-HARIS . PRIYONO . SISWANTO . SUMARYONO Afandi, Dadang Agustin Sri MULYATNI Akhirta Atikana Aksa, Annisa Aulia Alfero Putra Iryanto Anggia Prasetyoputri Annisa A Aksa Asmini Budiani Asmini BUDIANI Asmini Budiani Asmini Budiani Asmini Budiani Asmini BUDIANI1 Barahima Abbas Christy Dewi Sukma Dini Astika Sari Dini Astika Sari Dini Astika Sari Ernayunita Ernayunita Ernayunita, Ernayunita Galuh Wening PERMATASARI Galuh Wening Permatasari Galuh Wening PERMATASARI Happy WIDIASTUTI Hayati MINARSIH Hayati Minarsih Hayati Minarsih Hayati Minarsih Imam Bagus Nugroho Imron Riyadi Imron Riyadi Imron RIYADI Imron Riyadi Indra Syahputra Inez Palupi Irfan MARTIANSYAH Irfan Martiansyah Iskandar, Hayati Minarsih Jembar PAMBUDI Julie LECLERCQ Lailia Zubaidah Larasati D Mahardhika Lutfi Anggadhania Mardhika, Larasati Dena MARTIANSYAH, Irfan Masna Maya Sinta Masna Maya Sinta Mose, Windi Muhammad Farrel Ewaldo Mukmin, Restu Prasetya Nurul Khumaida Nurul Khumaida Pambudi, Jembar Pascal MONTORO Pascal MONTORO Pascal MONTORO PERMATASARI, Galuh Wening pri yono Radite TISTAMA Ratih Asmana Ningrum Rizka Tamania Saptari Rizka Tamania SAPTARI Rizka Tamania Saptari Rokhana Faizah Rokhana Faizah Sapto Nugroho Hadi Sapto Nugroho Hadi Sari, Dini Astika Sekar WOELAN Sri Wening Sri Wening Sudarsono Sudarsono Suhandono, Sonny Sustiprijatno Sustiprijatno, Sustiprijatno TATI KRISTIANTI Turhadi Turhadi Vivi Restu Raharti Y.M.M Anita Nugraheni Yuli Setiawati Yuli Setiawati Yuli Setiawati, Yuli

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