1. ISOLASI DAN DETEKSI GEN HOMOGLUTATION SINTETASE BINTIL AKAR LEGUM LIAR DENGAN TEKNIK PCR ISOLATION AND DETECTION OF HOMOGLUTHATION SINTETASE GENE ON ROOT NODULE OF WILD TYPE LEGUMES USING PCR TECHNIQUE

Abstract

ABSTRAK

Rendahnya kadar homoglutation akibat tidak aktifnya enzim homoglutation sintetase, menyebabkan terhambatnya fiksasi nitrogen bintil akar tanaman legum pada kondisi defisit air. Artikel ini melaporkan proses isolasi dan subklon gen homoglutation sintetase (hGSHS) pada bintil akar beberapa jenis legum liar dengan menggunakan pendekatan teknik PCR (Polymerase Chain Reaction).  Percobaan diawali dengan isolasi DNA genom bintil akar, yang dilanjutkan dengan deteksi gen hGSHS menggunakan teknik PCR. Fragmen DNA produk PCR sesuai panjang basa gen hGSHS (654 bp), dimurnikan dengan kolom PGX (kit invitrogen), dan disubklon pada vector TOPO. Kebenaran keberadaan gen target pada vector dilakukan analisis koloni PCR, dan sequen gen target menggunakan DNA sequencer. Hasil penelitian menunjukkan bahwa gen hGSHS telah berhasil disubklon pada vector TOPO, dengan arah yang sesuai dengan konstruksi yang ditetapkan.

ABSTRACT

Low homogluthatione content as a result of inactively homogluthation synthetase, inhibits nitrogen fixation by root nodules of legumes grown under water deficit condition. This article reports the isolation and subcloning process of homogluthatione synthetase gene (hGSHS) on root nodules of wild type legumes using PCR technique. The experiment was initiated by the isolation of DNA genom from root nodules of several legumes. Then, hGSHS gene was detected using PCR. Fragment DNA as long as 654 bp (hGSHS gene) was purified using PGX column (Invitrogen kit), and subcloned into TOPO cloning vector. The existance of gene target in the vector, it was confirmed by colony PCR and DNA sequent analysis. The results shown that hGSHS gen was successfully subcloned on TOPO cloning vector whith right direction according to construct has been regulated.