Construction and Transformation of HVA1 Gene Expression Vector into Indonesian Elite Rice Varieties

Abstract

of drought in rice. HVA1 is one of the Late Embryogenesis Abundant (LEA) protein group that plays a role on cell protection during stresses. A study was done with an objective to construct a plasmid vector expressing HVA1 and to transform it into Indonesian elite rice varieties. Materials used in the study were plasmid pBY520 (source of HVA1; intermediate plasmid pRP9; plasmid pAY560326 (backbone); restriction enzymes BamHI, HindIII, XhoI, and SpeI; T4 DNA ligase, and gel DNA extraction kit. Methods used were standard procedure for plasmid vector construction and molecular biology. Step I: the pBY520 and pRP9 were cut with BamHI and HindIII, and electrophorated with 1% agarose gel. DNA fragments of HVA1 and pRP9 were purified, ligated with T4 DNA ligase, and transformed into Escherichia coli DH5-α by heat shock. E. coli were grown onto solid medium (+ kanamycin 100 mg/l). A new plasmid DNA was isolated from single colony culture of the bacteria, confirmed, and named pRP9_HVA1. Step II: DNA of pRP9_HVA1 and pAY560326 were cut with XhoI dan SpeI enzymes, purified, and ligated. The next procedure was similar to step I, and the resulted plasmid was confirmed by PCR and digestion with XhoI dan SpeI enzymes, and named pAY_HVA1. Step III: pAY_HVA1 was first transformed into Agrobacterium EHA-105 and then into rive varieties Ciherang and Inpari 6 using the early infection of scutellum transformation method. Nine transgenic rice lines that positively contain HVA1 were obtained.