Download Construction of Expression Cassettes for Gene Silencing of

Construction of Expression Cassettes for Gene Silencing of Stachyose
Synthase from Soybean (Glycine max L. Meril), via RNA Interference
Soares, A.P.G.1; Queiroz, L.N.1; Rezende, S.T.1; Barros, E.G.2; Guimarães, V.M.1
1
Departamento de Bioquímica e Biologia Molecular, BIOAGRO, UFV, MG, Brazil;
2
Departamento de Biologia Geral, BIOAGRO, UFV, MG, Brazil
Soybeans are an excellent source of protein, fiber and polyunsaturated oils,
however there are factors that limit their consumption as food, such as raffinose
oligosaccharides (ROs), which are responsible for gastrointestinal disorders. The
silencing of specific genes in the biosynthesis of ROs by RNA interference may
result in soybean varieties with low content of ROs. The aim of this study was to
construct expression cassettes, targetted at silencing the gene encoding the
enzyme stachyose synthase (STS) in soybean via RNA interference. After
extraction of total RNA from soybean seeds, cDNA was synthesized by RT-PCR.
The STS gene, 2.6 kb, was amplified by PCR using specific primers, cloned and
sequenced. Internal fragments, corresponding to the sense and antisense
fragments, 368 bp, was amplified using specific internal primers. These fragments
were cloned into the pGEM-T Easy vector and sequenced. The fragments were
then cloned into the expression vector pKANNIBAL flanked by the constitutive
promoter (CaMV35S) or by the seed-specific beta-conglycinin promoter; the OCS
terminator region, and spaced by the PDK intron. The cassettes obtained were
transferred to the vector pCAMBIA 3301, for insertion into the genome of soybean
via Agrobacterium tumefaciens.
Word Keys: gene silencing, RNA interference, stachyose synthase
Supported by: FAPEMIG and CAPES
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