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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.

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.

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
Menara Perkebunan Vol. 83 No. 2: 83 (2), 2015
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v83i2.4

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.

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
Menara Perkebunan Vol. 84 No. 1 (2016): 84 (1), 2016
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

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.

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