Introduksi Konstruk Over-Ekspresi Kandidat Gen OsWRKY76 melalui Agrobacterium tumefaciens pada Tanaman Padi Nipponbare

Apriana, Aniversari; Sisharmini, Atmitri; Enggarini, Wening; Sudarsono, Sudarsono; Khumaida, Nurul; Trijatmiko, Kurniawan Rudi

doi:10.21082/jbio.v7n1.2011.p19-27

Cited by 4 publications

(5 citation statements)

References 4 publications

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“…dan sekitar 1.600 bp yang merupakan fragmen gen kandidat AlaAT. Hasil validasi ini sejalan dengan penelitian sebelumnya, analisis pemotongan plasmid rekombinan yang terkonfirmasi positif menghasilkan fragmen berukuran 3.000 bp yang merupakan plasmid pGEM®-T Easy dan 1.200 bp yang merupakan fragmen gen kandidat OsWRKY76 (Apriana et al 2011).…”

Section: Penyisipan Fragmen Gen Alaat Ke Dalam Vektor Kloning Dan Verunclassified

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

Sisharmini¹,

Apriana²,

Santoso³

et al. 2020

J. AgroBiogen

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Overexpression of alanine aminotransferase (AlaAT) gene can improve nitrogen use efficiency (NUE) in plants. The previous isolated AlaAT genes cannot be freely applied to generate NUE plants due to IPR restriction. Therefore, isolation of the gene from targeted monocot and dicot plants is necessary. The objectives of this study were to isolate AlaAT genes from barley, foxtail millet, cucumber, and tomato and analyze their homology to other isolated AlaAT genes in sequence databases (gene bank). Total RNA was isolated from roots of barley, foxtail millet, cucumber, and tomato, and then converted into cDNA using reverse transcription method. The cDNA was then cloned into pGEM®-T Easy plasmid and the verified clones were sequenced. The amino acid sequences were analyzed for their homologies using Clustal Omega software and phylogenetic tree was constructed. The results showed that the amino acids of AlaAT gene from barley was different from AlaAT genes of tomato and cucumber with homology level less than 80%. Phylogenetic tree predicted that AlaAT genes clustered into three groups with AlaAT genes of foxtail millet and barley clustered in one group together with other monocots in group I. AlaAT genes derived from dicots clustered into two groups in which AlaAT gene of tomato clustered in group II, while that of cucumber was in group III. The identity differences of AlaAT gene of tomato and that of cucumber as well as the estimates of the same enzymatic functions can open up enormous opportunities in genetic engineering research for the development of NUE rice.

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Section: Penyisipan Fragmen Gen Alaat Ke Dalam Vektor Kloning Dan Verunclassified

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

Sisharmini¹,

Apriana²,

Santoso³

et al. 2020

J. AgroBiogen

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“…Padi telah menjadi tanaman monokotil rujukan atau tanaman model yang disebabkan berlimpahnya informasi molekuler (Sakata et al 2000), efisiensi transformasi yang relatif tinggi (Tyagi dan Mohanty 2000), dan ukuran genom yang kecil (430 Mb) (Arumuganathan dan Earle 1991). Nipponbare merupakan salah satu varietas model yang memiliki efisiensi transformasi yang cukup tinggi (Apriana et al 2011;Enggarini et al 2017;Windiastri et al 2018) dan informasi genetik yang lengkap (Matsumoto et al 2016…”

Section: Pendahuluanunclassified

“…Nilai efisiensi ini sangat mirip dengan hasil penelitian transformasi yang dilaporkan oleh Enggarini et al (2017) sebesar 11,9% dengan metode transformasi yang sama. Bahkan, penggunaan A. tumefaciens strain LBA4404 pada penelitian ini menghasilkan efisiensi transformasi lebih tinggi dibanding dengan penggunaan strain Agrobacterium lainnya seperti strain Agl-1 dan strain EHA 105 yang berturut-turut hanya mencapai 9,6% dan 2,6% (Apriana et al 2011). Efisiensi transformasi sangat dipengaruhi oleh beberapa faktor, di antaranya strain Agrobacterium, genotipe tanaman, jenis eksplan, vektor plasmid, komposisi media kultur, dan pengurangan tekanan infeksi Agrobacterium setelah kokultivasi (Opabode 2006).…”

Section: Hasil Dan Pembahasan Struktur Sekuen Gen Lccsp Hasil Modifikasiunclassified

Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method

et al. 2020

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Cold shock protein (Csp) is an essential bacterial protein for increasing abiotic stress tolerance, especially cold stress. Several studies discovered that overexpression of the gene successfully improves the tolerances of several types of plant not only under cold stress, but also other abiotic stresses, e.g. hot and drought conditions. The objectives of this study were to construct a binary vector containing the LcCsp gene modified from Lactobacillus casei and transform the gene into Nipponbare rice genome. The native LcCsp gene sequence, however, has low GC content (46.7%) while rice as transformation target plant has 52% GC content. The native LcCsp gene sequence, therefore, was optimized to the level close to 52.7% similar to GC content of the rice genome. This LcCsp gene was synthesized by using DNA printing technology (gBlocks® Gene Fragments Entry, IDT). The synthetic LcCsp gene was successfully inserted into the pCAMBIA1300-int binary vector driven by Ubiquitin1 promoter and NOS terminator. The T-DNA cassette was successfully transformed into Nipponbare rice genome by Agrobacterium tumefaciens strain LBA4404 using immature embryo transformation protocol. A total of 51 T0 Nipponbare lines survived from hygromycin selections and 21 lines were successfully acclimatized. Molecular analysis of the candidate lines showed that all Nipponbare transgenic putative lines contain the LcCsp gene demonstrating high transformation efficiency of 11.8%. The rice lines resulted from this study should be further analyzed and might be useful for developing rice transgenic lines tolerance to heat, drought, or saline stress condition.

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“…DNA kemudian digunakan sebagai cetakan untuk analisis keberadaan gen hpt dan bar. Deteksi gen hpt dilakukan dengan metode Apriana et al (2011), sedangkan deteksi gen bar dilakukan dengan metode Sisharmini et al (2009). Komposisi komponen PCR untuk reaksi volume 20 µl adalah: 2,0 µl bufer PCR 10× (Tris HCl 100 mM, KCl 500 mM [pH 8,3]), 1,2 µl MgCl 2 50 mM, 0,4 µl dNTP mix 10 mM, 1 µl setiap primer (10 µM), 1 unit Taq DNA polimerase (5 unit/µl), dan 2 µl DNA cetakan.…”

Section: Kestabilan Posisi Elemen Ds Pada Padi Transgenik Penanda Aktunclassified

Evaluasi Toleran Kekeringan dan Analisis Kestabilan Padi Transgenik Penanda Aktivasi cv. Nipponbare

Ma’sumah¹,

Santoso²,

Trijatmiko³

2018

J. AgroBiogen

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Drought stress is one of the important limiting factors in increasing rice production in Indonesia. Development of rice varieties with increased tolerance to drought is needed to meet the rice production challenge. Some transgenic Nipponbare rice lines that carry activation tag have been generated from the previous study. The purpose of this study was to evaluate the root penetration ability and the stability of Ds element in the T 1 generation of activation tag rice lines. Materials used in the study were T 1 seeds of 47 transgenic lines, drought tolerant check varieties (Cabacu and IRAT112), and sensitive check varieties (IR64 and wild type Nipponbare). T 1 seeds were prescreened by germination in Basta herbicide solution to eliminate T 1 individuals that did not carry activation tag element. Root penetration ability was evaluated using wax-petrolatum layers as a proxy for compacted soil layers. The presence of bar gene and the absence of hpt gene as detected by PCR were used to identify T 1 stable mutants. Out of 47 transgenic lines tested, 38 lines showed better root penetration ability than non transformed Nipponbare. PCR analysis identified four stable mutants, namely M-Nip-12.12, M-Nip-19.8, M-Nip-19.9, and M-Nip-20.13. One stable mutant, M-Nip-20.13, showed better root penetration ability than tolerant check varieties. This mutant is a good candidate for isolation of drought tolerance gene. Keywords:Rice, transposon mutagenesis, activation tagging, drought tolerance. ABSTRAKCekaman kekeringan merupakan salah satu faktor pembatas penting dalam peningkatan produksi padi di Indonesia. Perakitan varietas unggul padi toleran kekeringan diperlukan untuk menjawab tantangan tersebut. Beberapa galur padi Nipponbare transgenik yang membawa penanda aktivasi telah dihasilkan pada penelitian sebelumnya. Tujuan penelitian ini adalah mengevaluasi daya tembus akar dan stabilitas elemen Ds pada galur-galur penanda aktivasi generasi T 1 . Bahan yang digunakan dalam penelitian ini adalah benih T 1 47 galur transgenik, varietas cek toleran kekeringan (Cabacu dan IRAT112), dan varietas cek peka (IR64 dan Nipponbare tipe liar). Benih T 1 ditapis terlebih dahulu dengan perkecambahan pada larutan herbisida Basta untuk mengeliminasi individu yang tidak membawa elemen penanda aktivasi. Daya tembus akar dievaluasi menggunakan lapisan lilin sebagai tiruan lapisan tanah yang padat dan keras (hardpans). Keberadaan gen bar dan ketiadaan gen hpt yang dideteksi dengan PCR digunakan untuk mengidentifikasi mutan-mutan stabil. Dari 47 galur yang diuji, 38 galur menunjukkan daya tembus akar yang lebih baik daripada Nipponbare non transforman. Analisis PCR mengidentifikasi empat mutan stabil, yaitu M-Nip-12.12, M-Nip-19.8, M-Nip-19.9, dan M-Nip-20.13. Satu mutan stabil, M-Nip-20.13, menunjukkan daya tembus akar yang lebih baik daripada varietas cek toleran. Mutan ini menjadi kandidat yang baik untuk isolasi gen toleran kekeringan.Kata kunci: Padi, mutagenesis transposon, penanda aktivasi, toleransi kekeringan.Hak Cipta © 2016, BB B...

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Introduksi Konstruk Over-Ekspresi Kandidat Gen OsWRKY76 melalui Agrobacterium tumefaciens pada Tanaman Padi Nipponbare

Cited by 4 publications

References 4 publications

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method

Evaluasi Toleran Kekeringan dan Analisis Kestabilan Padi Transgenik Penanda Aktivasi cv. Nipponbare

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