Figure 4.

Levels of mRNA and PLDα protein in the immature and mature cotyledons from SW progeny and the background cultivar ‘Fayette’. The SW progeny line, SW-N, was characterized by PCR and Southern blot analysis as a homozygous, non-transgenic line, segregated from the T₂ progeny of SW. (a); RNA blot analysis of steady-state levels for HPT in transgenic soybean lines and background cultivar. Total RNA are from the immature soybean seeds that were background cultivar (Fayette), null transgenic line (SW-N), and PLDα suppressed line (SW). (b); Quantitative PCR analysis of PLDαβ-like, and γ-like mRNAs from immature and mature cotyledon respectively. The levels of endogenous of PLDαβ-like, and γ-like mRNAs were measured by real-time PCR from null transgenic line (SW-N) and PLDα suppressed line (SW). Changes in PLD mRNA transcription were represented suppression compared to the level of PLD mRNA transcription in Fayette. PCR primers of soybean PLDβ-like and γ-like were designed from soybean EST clones, gi:21888889 and gi:17401471, which showed sequence similarity to 5’UTR region in AtPLDβ and AtPLDγ respectively. (c); Changes in PLDα activities in immature and mature cotyledons from null transgenic line (SW-N) and PLDα suppressed line (SW). The PLDα enzyme activity was expressed as nmoles of phosphatidylcholine min⁻¹ mg⁻¹ total protein. Error bars indicates ± SE with three independent measurements. (d); Immunoblot analysis of cytosolic protein from immature and mature cotyledon with polyclonal anti-AtPLDα antibody. Proteins (40 µg per lane) were separated in SDS-PAGE in 12% polyacylamide gel and PLDα protein was visualized by alkaline phosphatase.