SANTOSO,, D. and CHAIDAMSARI, T. and BUDIANI, A. and MINARSIH, H and Utomo, Setyo Dwi and Siswanto, Siswanto (2000) 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.). Menara Perkebunan, 68 (2).

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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. KEYWORDS Gene expression, recombinant DNA, transgenic tobacc

Item Type:	Article
Subjects:	S Agriculture > SB Plant culture
Divisions:	Fakultas Pertanian (FP) > Prodi Agronomi dan Hortikultura
Depositing User:	Prof. Setyo Utomo
Date Deposited:	25 Oct 2016 01:11
Last Modified:	25 Oct 2016 01:11
URI:	http://repository.lppm.unila.ac.id/id/eprint/899

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