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

Sri Koerniati, Hani Widhianata

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.


Keywords


Vector construction; over-expression; osmoprotectant gene HVA1; drought tolerant rice.

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DOI: http://dx.doi.org/10.21082/jbio.v8n2.2012.p54-61

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P-ISSN : 1907-1094
E-ISSN : 2549-1547


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