Showing papers by "Suharsono Suharsono published in 2009"

ISOLATION AND CLONING OF cDNA OF GENE ENCODING FOR METALLOTHIONEIN TYPE 2 FROM MELASTOMA AFFINE

Abstract: Metallothionein is an important protein for detoxifying heavy metal ions. Th is research was conducted to isolate and clone cDNA of gene encoding for metallothionein type 2 from Melastoma affi ne. Total RNA was isolated from young leaves. Total cDNA was synthesized from the total RNA by reverse transcription. Th e MaMt2 cDNA was successfully isolated by PCR technique. Th e MaMt2 cDNA was inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into Escherichia coli DH5α. DNA sequencing analysis showed that this cDNA is full length consisting of 246 pb encoding 81 amino acid residues. Th is cDNA is identical to mRNA of AtMt2 from Arabidopsis thaliana. It does not contain any restriction sites found in the cloning sites of pGEM-T Easy. Th e deduced protein of MaMT2 contains 14 cysteine residues distributed in the Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys motifs.

Isolation and Cloning of cDNA of Gene Encoding for Metallothionein Type 2 from Soybean [Glycine max (L.) (Merrill)] cv Slamet

Abstract: Metallothionein has an important role in the detoxification of metal ions. It has a low molecular weight and contains cysteine-rich residue. The objective of this research is to isolate and clone the cDNA of gene encoding for metallothionein from soybean [Glycine max (L.) (Merrill)] cv Slamet (GmMt2). We had successfully isolated total RNA by reverse transcription and synthesized total cDNA from total RNA as template. cDNA of GmMt2 had been isolated from total cDNA by PCR. It was successfully inserted into pGEM-T Easy plasmid, and the recombinant plasmids were introduced into Escherichia coli strain DH5I±. Sequence analysis by using T7 and SP6 primers showed that the length of PCR-isolated fragment was 257 bp containing 246 bp completed sequence of Mt2 cDNA encoding for 81 amino acids. Enzyme restriction analysis showed that GmMt2 did not contain any restriction sites found in the multi cloning sites of pGEM-T easy. Nucleotide and amino acid alignment analysis using BLAST program showed that GmMt2 was similar with completed cDNA of AtMt2A from Arabidopsis thaliana (L.) Heynh. Amino acid sequence analysis showed that the motifs of Cys sequence of GmMT2 are Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys.Key words: soybean, metallothionein, cDNA, isolation, cloning, cysteine.