Genetic transformation of Nicotiana benthamiana L. and soybean with MaMt2 Gene Encoding Metallothionein Type II from Melastoma malabathricum L.

Abstract

The low pH and high concentration of aluminum (Al) are major limiting factors for crops production. The capability of crops to cope those stresses can be improved by expressing genes controlling adaptation to low pH and high concentration of Al. One of these genes was MaMt2 encoding for metallothionein type II which isolated from Melastoma malabathricum. The objectives of the research were 1) to construct overexpression vector of MaMt2 gene with pIG6 plasmid, 2) to transform Nicotiana benthamiana and soybean with MaMt2 gene, and to analyze the transgenic plants. The MaMt2 gene were successfully fused with the strong promoter pUbiquitin in the pIG6 plasmid, and the recombinant plasmid was introduced into Escherichia coli DH5α by electroporation method. The pIG6-MaMt2 recombinant vector was successfully introduced into A. tumefaciens LBA4404 by triparental mating method (TPM). Genetic transformation was performed by co-cultivating A. tumefaciens LBA4404-pIG6-MaMt2 with N. benthamiana leaf explants and half seed explants of soybean cv. Lumut. The T0 and T1 generations of transgenic plants were already obtained. The integration of MaMt2 transgene into the genom of T0 transgenic plants was confirmed by PCR. Segregation analysis in the T1 generation of N. benthamiana showed that hpt gene was inherited to the offspring in Mendelian pattern and all samples of transgenic plants were heterozygote containing one functional hpt gene. Transformation of cv. Lumut half seed explants with MaMt2 gene was succesful, based on PCR by UbiF and NosTR primer combination. PCR analysis of T1 generation of transgenic soybean showed that MaMt2 gene was transmitted into the offspring indicating that this transgene is integrated in the genome of transgenic soybean plants.

Cekaman pH rendah dan kandungan aluminium yang tinggi pada tanah masam, khususnya tanah Ultisol, merupakan faktor-faktor pembatas produksi dan kualitas tanaman pangan. Kemampuan beberapa tanaman pangan penting dalam mengatasi toksisitas logam dapat ditingkatkan dengan cara mengekspresikan gen-gen yang terkait dengan toleransi pH rendah dan aluminium (Al) tinggi. Melastoma malabathricum merupakan salah satu tanaman yang mampu hidup di tanah ber- pH rendah dan kelarutan Al tinggi. Sejumlah gen yang ekspresinya diinduksi oleh cekaman pH rendah dan aluminium tinggi pada M. malabathricum sebagian sudah berhasil diisolasi dan dikarakterisasi. Salah satu gen tersebut adalah gen MaMt2 yang menyandi metallothionein tipe II. Protein ini banyak mengandung sistein yang mampu mengikat berbagai macam logam. Penelitian ini bertujuan untuk mengkonstruksi vektor ekspresi berlebih gen MaMt2 menggunakan plasmid pIG6, mentransformasi tanaman Nicotiana benthamiana dan kedelai dengan gen MaMt2, dan menganalisis tanaman transgenik secara molekular.