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Overexpression of chitinase gene with a GC-rich synthetic enhancer in tobacco plant (Nicotiana tabacum L.) Overekspresi gen kitinase dengan enhancer sintetis kaya GC pada tanaman tembakau (Nicotiana tabacum L.) D SANTOSO; T CHAIDAMSARI; A BUDIANI; H MINARSIH; S DWI UTOMO; . SISWANTO
E-Journal Menara Perkebunan Vol 68, No 2: Desember 2000
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (264.272 KB) | DOI: 10.22302/iribb.jur.mp.v68i2.139

Abstract

Ringkasan Perakitan tanaman perkebunan toleran terhadap serangan cendawan patogenik  dilakukan dengan mengoverekspresikan gen penyandi kitinase. Untuk itu elemen DNA peningkat ekspresi (enhancer E52) yang berupa oligonukleotida 52 pb dan kaya kandungan basa purin (GC) disisipkan di ujung 5’ konstruk 35S-chi. Penyisipan E52 tersebut dilakukan secara lebih terarah pada situs ganda HindIII-SalI dari MCS pCAMBIA2301. Melalui situs HindIII yang terletak tepat di ujung 3’ E52, konstruk 35S-chi kemudian disambungkan dengan E52 pada pCAMBIA tersebut. Transformasi DNA rekombinan ke dalam sel tembakau dikerjakan melalui perantaraan Agrobacterium tumefaciens LBA4404. Sel tanaman transgenik     diseleksi    dan  diregenrasi dalam media  yang    mengandung  3%  sukrosa, 0,5 mg/L diregenerasikan pada media   MS   padat  benzilaminopurin   (BAP)  dan  50 mg/L kanamisin. Pada media ini tunas transgenik    tembakau   mulai   terbentuk    setelah 5 minggu penanaman. Analisis  tingkat aktivitas enzimatis menunjukkan bahwa aktivitas kitinase pada tembakau transgenik 40 hingga 80 kali lebih tinggi daripada non-transgenik. Pengujian hibridisasi protein menggunakan antibodi anti-kitinase, dot blot dan western blot, membuktikan bahwa enhancer tersebut dapat meningkatkan ekspresi transgen chi pada tanaman tembakau.Summary Development of estate crops tolerant to pathogenic fungi is conducted by overexpressing chi gene. For this purpose, a synthetic enhancer consisting of 52 base pairs and GC-rich was inserted at immediate 5’ end of a 35S-chi cassette. Insertion of the E52 was directed at HindIII-SalI restriction sites of  the pCAMBIA2301 MCS.  With HindIII restriction site located just after the 3’ end of the E52 sequence, the 35S-chi construct was then ligated with the E52 of the pCAMBIA. Transformation of the resulting recombinant DNA into tobacco cells was mediated by Agrobacterium tumefaciens LBA4404. The transgenic cells were selected and regenerated on a solid MS medium supplemented with 3% sucrose, 0.5 mg/L benzylamino purine (BAP) and 50 mg/L kanamycin. Tobacco   shoots   were   initiated  after 5 weeks inoculation on the selection media. Enzymatic analysis demonstrated that chitinase activity of transgenic tobacco was 40 to 80 folds higher than that of the control plant. Analysis of  enzymatic activity using hybridization with anti-chitinase antibody indicated that the level of chitinase activity in the transgenic tobacco carrying the enhancer is higher than that  without enhancer. These data suggest that the enhancer improved the expression of chi transgene in tobacco. 
Isolation and characterization of protein differentially expressed during oil palm mesocarp development Isolasi dan karakteristik protein terekpresi secara diferensial selama perkembangan mesokarp pada kelapa sawit A BUDIANI; D SANTOSO; H ASWINDINNOOR; A SUWANTO; S SUDIATSO
E-Journal Menara Perkebunan Vol 70, No 1: Juni 2002
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (273.136 KB) | DOI: 10.22302/iribb.jur.mp.v70i1.130

Abstract

RingkasanPada kelapa sawit, mesokarp merupakan jaringan yang lebih dikhususkan untuk mensintesis minyak. Akumulasi minyak pada jaringan ini terjadi selama perkembangan buah. Beberapa enzim yang terlibat dalam biosintesis minyak tampaknya disintesis hanya pada periode tertentu dari biosintesis minyak. Sedangkan protein regulator diduga ada pada saat minyak mulai disintesis atau beberapa saat sebelumnya. Sebagai bagian dari usaha mengklon gen kunci untuk biosintesis minyak, penelitian ini bertujuan mengidentifikasi dan mengisolasi protein yang terekspresi secara diferensial sesuai perkembangan buah. Sebagai bahan penelitian digunakan jaringan mesokarp dari berbagai umur buah sawit. Untuk setiap fase perkembangan buah dilakukan analisis kandungan minyak dan protein total. Elektroforesis gel poliakrilamid-SDS (SDS – PAGE) dan elektroforesis dua dimensi (2-D) digunakan untuk mempelajari dan mendeteksi adanya pita protein spesifik yang terekspresi secara diferensial sejalan dengan peningkatan kandungan minyaknya. Hasil penelitian menunjukkan bahwa minyak mulai aktif disintesis pada saat buah berumur 17 minggu setelah antesis. Konsentrasi protein total tidak meningkat sejalan dengan peningkatan kandungan minyaknya. Dari hasil SDS-PAGE terdeteksi dua protein, yaitu protein dengan berat molekul (BM) 31,0 kDa dan 34,3 kDa yang meningkat ekspresinya pada awal dan menjelang periode aktif sintesis minyak. Analisis lebih lanjut dengan elektroforesis 2-D menunjukkan bahwa protein 31,0 kDa terdiri dari dua protein dengan pI 4,64 dan pI 4,95, sedangkan protein 34,3 kDa merupakan protein tunggal dengan pI 4,56. Sikuensing secara parsial kedua protein tersebut menunjukkan adanya dua polipeptida dari protein 31,0 kDa yang mempunyai homologi tinggi dengan subunit biotin karboksilase ht-ACCase, dan empat polipeptida yang mempunyai homologi dengan enoilACP reduktase. Sedangkan protein 34,3 kDa mempunyai homologi dengan gliseraldehida 3-fosfat dehidrogenase. SummaryIn oil palm, mesocarp is tissue specialized for oil synthesis. Accumulation of oil in this tissue occurs during fruit development. It is likely that some enzymes involved in oil biosynthesis are synthesized only in a certain period of oil biosynthesis, while regulatory proteins may present at the beginning or right before the period of active oil synthesis. As a part of research work on cloning of gene encoding key enzymes for oil biosynthesis in palm mesocarp, this research was aimed to identify and isolate proteins differentially expressed during fruit development. Mesocarps from different developmental stage of fruit were used for analysis of oil content and protein concentra-tion. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and two dimentional (2-D) electrophoresis were used to study and detect specific protein bands differentially expressed during fruit development. It was shown that oil synthesis was started at 17 weeks after anthesis (WAA). There was no correlation between concentrations of total protein with oil content during mesocarp development. From the SDS-PAGE, two protein of 31.0 kDa and 34.3 kDa were detected that their expression increased at the beginning and just before the period of active oil biosynthesis respectively. Further analysis with 2-D electrophoresis showed that 31.0 kDa-protein consist of two proteins, with pI 4,64 and pI 4,95, while 34.3 kDa protein is a single protein with pI 4,56. Partial amino acid sequencing data of the 31.0 kD protein showed that two polypeptides highly homologous with ht-ACCase biotin carboxylase subunit and four polypeptides homologuus with enoyl-ACP reductase, whereas 34.3 kD protein showed homology with glyceraldehyde-3 phosphate dehydrogenase.
Isolation and characterization of protein differentially expressed during oil palm mesocarp development Isolasi dan karakteristik protein terekpresi secara diferensial selama perkembangan mesokarp pada kelapa sawit A BUDIANI; D SANTOSO; H ASWINDINNOOR; A SUWANTO; S SUDIATSO
Menara Perkebunan Vol. 70 No. 1: 70 (1), 2002
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v70i1.130

Abstract

RingkasanPada kelapa sawit, mesokarp merupakan jaringan yang lebih dikhususkan untuk mensintesis minyak. Akumulasi minyak pada jaringan ini terjadi selama perkembangan buah. Beberapa enzim yang terlibat dalam biosintesis minyak tampaknya disintesis hanya pada periode tertentu dari biosintesis minyak. Sedangkan protein regulator diduga ada pada saat minyak mulai disintesis atau beberapa saat sebelumnya. Sebagai bagian dari usaha mengklon gen kunci untuk biosintesis minyak, penelitian ini bertujuan mengidentifikasi dan mengisolasi protein yang terekspresi secara diferensial sesuai perkembangan buah. Sebagai bahan penelitian digunakan jaringan mesokarp dari berbagai umur buah sawit. Untuk setiap fase perkembangan buah dilakukan analisis kandungan minyak dan protein total. Elektroforesis gel poliakrilamid-SDS (SDS – PAGE) dan elektroforesis dua dimensi (2-D) digunakan untuk mempelajari dan mendeteksi adanya pita protein spesifik yang terekspresi secara diferensial sejalan dengan peningkatan kandungan minyaknya. Hasil penelitian menunjukkan bahwa minyak mulai aktif disintesis pada saat buah berumur 17 minggu setelah antesis. Konsentrasi protein total tidak meningkat sejalan dengan peningkatan kandungan minyaknya. Dari hasil SDS-PAGE terdeteksi dua protein, yaitu protein dengan berat molekul (BM) 31,0 kDa dan 34,3 kDa yang meningkat ekspresinya pada awal dan menjelang periode aktif sintesis minyak. Analisis lebih lanjut dengan elektroforesis 2-D menunjukkan bahwa protein 31,0 kDa terdiri dari dua protein dengan pI 4,64 dan pI 4,95, sedangkan protein 34,3 kDa merupakan protein tunggal dengan pI 4,56. Sikuensing secara parsial kedua protein tersebut menunjukkan adanya dua polipeptida dari protein 31,0 kDa yang mempunyai homologi tinggi dengan subunit biotin karboksilase ht-ACCase, dan empat polipeptida yang mempunyai homologi dengan enoilACP reduktase. Sedangkan protein 34,3 kDa mempunyai homologi dengan gliseraldehida 3-fosfat dehidrogenase. SummaryIn oil palm, mesocarp is tissue specialized for oil synthesis. Accumulation of oil in this tissue occurs during fruit development. It is likely that some enzymes involved in oil biosynthesis are synthesized only in a certain period of oil biosynthesis, while regulatory proteins may present at the beginning or right before the period of active oil synthesis. As a part of research work on cloning of gene encoding key enzymes for oil biosynthesis in palm mesocarp, this research was aimed to identify and isolate proteins differentially expressed during fruit development. Mesocarps from different developmental stage of fruit were used for analysis of oil content and protein concentra-tion. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and two dimentional (2-D) electrophoresis were used to study and detect specific protein bands differentially expressed during fruit development. It was shown that oil synthesis was started at 17 weeks after anthesis (WAA). There was no correlation between concentrations of total protein with oil content during mesocarp development. From the SDS-PAGE, two protein of 31.0 kDa and 34.3 kDa were detected that their expression increased at the beginning and just before the period of active oil biosynthesis respectively. Further analysis with 2-D electrophoresis showed that 31.0 kDa-protein consist of two proteins, with pI 4,64 and pI 4,95, while 34.3 kDa protein is a single protein with pI 4,56. Partial amino acid sequencing data of the 31.0 kD protein showed that two polypeptides highly homologous with ht-ACCase biotin carboxylase subunit and four polypeptides homologuus with enoyl-ACP reductase, whereas 34.3 kD protein showed homology with glyceraldehyde-3 phosphate dehydrogenase.
Overexpression of chitinase gene with a GC-rich synthetic enhancer in tobacco plant (Nicotiana tabacum L.) Overekspresi gen kitinase dengan enhancer sintetis kaya GC pada tanaman tembakau (Nicotiana tabacum L.) D SANTOSO; T CHAIDAMSARI; A BUDIANI; H MINARSIH; S DWI UTOMO; . SISWANTO
Menara Perkebunan Vol. 68 No. 2: 68 (2), 2000
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v68i2.139

Abstract

Ringkasan Perakitan tanaman perkebunan toleran terhadap serangan cendawan patogenik  dilakukan dengan mengoverekspresikan gen penyandi kitinase. Untuk itu elemen DNA peningkat ekspresi (enhancer E52) yang berupa oligonukleotida 52 pb dan kaya kandungan basa purin (GC) disisipkan di ujung 5’ konstruk 35S-chi. Penyisipan E52 tersebut dilakukan secara lebih terarah pada situs ganda HindIII-SalI dari MCS pCAMBIA2301. Melalui situs HindIII yang terletak tepat di ujung 3’ E52, konstruk 35S-chi kemudian disambungkan dengan E52 pada pCAMBIA tersebut. Transformasi DNA rekombinan ke dalam sel tembakau dikerjakan melalui perantaraan Agrobacterium tumefaciens LBA4404. Sel tanaman transgenik     diseleksi    dan  diregenrasi dalam media  yang    mengandung  3%  sukrosa, 0,5 mg/L diregenerasikan pada media   MS   padat  benzilaminopurin   (BAP)  dan  50 mg/L kanamisin. Pada media ini tunas transgenik    tembakau   mulai   terbentuk    setelah 5 minggu penanaman. Analisis  tingkat aktivitas enzimatis menunjukkan bahwa aktivitas kitinase pada tembakau transgenik 40 hingga 80 kali lebih tinggi daripada non-transgenik. Pengujian hibridisasi protein menggunakan antibodi anti-kitinase, dot blot dan western blot, membuktikan bahwa enhancer tersebut dapat meningkatkan ekspresi transgen chi pada tanaman tembakau.Summary Development of estate crops tolerant to pathogenic fungi is conducted by overexpressing chi gene. For this purpose, a synthetic enhancer consisting of 52 base pairs and GC-rich was inserted at immediate 5’ end of a 35S-chi cassette. Insertion of the E52 was directed at HindIII-SalI restriction sites of  the pCAMBIA2301 MCS.  With HindIII restriction site located just after the 3’ end of the E52 sequence, the 35S-chi construct was then ligated with the E52 of the pCAMBIA. Transformation of the resulting recombinant DNA into tobacco cells was mediated by Agrobacterium tumefaciens LBA4404. The transgenic cells were selected and regenerated on a solid MS medium supplemented with 3% sucrose, 0.5 mg/L benzylamino purine (BAP) and 50 mg/L kanamycin. Tobacco   shoots   were   initiated  after 5 weeks inoculation on the selection media. Enzymatic analysis demonstrated that chitinase activity of transgenic tobacco was 40 to 80 folds higher than that of the control plant. Analysis of  enzymatic activity using hybridization with anti-chitinase antibody indicated that the level of chitinase activity in the transgenic tobacco carrying the enhancer is higher than that  without enhancer. These data suggest that the enhancer improved the expression of chi transgene in tobacco.