Page 7 - Legume Genomics and Genetics

Legume Genomics and Genetics (online), 2010, Vol. 1, No.3, 11-17
http://lgg.sophiapublisher.com
dominantly in leaf among four tissues tested, and ex-
pressed at the lowest level in stem. In addition, com-
parative analysis among seeds over five development
stages showed that AhKASⅠ gene expressed in an
irregular course during seed development. It had high-
est expression at 45 DAP and the expression level at
65 DAP was also relatively high (Figure 5).
14
Figure 4 Expression analysis of AhKASⅠ gene in fo differentur
tissues
Figure 5 Expression analysis of AhKASⅠ gene in seed different
carbon–carbon bonds is a fundamental L. cultivar Huayu19)
d soil mixture (1:1), grown in a
ed from samples using the
to the manu-
at
developmental stages
2 Discussion
The formation of
biochemical reaction. A number of enzymes involved
in various biosynthetic pathways accomplish this reac-
tion by different means. Among them, one mechanism
is the Claisen condensation, a reaction catalyzed by β-
ketoacyl-ACP synthase (KAS) enzymes (Olsen et al.,
2001). In higher plants five types of KAS, namely,
KASⅠ, KASⅡ , KASⅢ, KASⅣ and mitochondrial
KAS, have been reported (Li et al., 2009). In the pre-
sent study, we isolated a KASⅠ orthologue in peanut
seedling. We investigated the homology of this gene,
the deduced protein’s active sites, physicochemical pro-
perties and its subcellular location through bioin-
formatics analysis. The results revealed that the amino
acid sequences of peanut AhKASⅠ shared high
sequence identity, 90.2% and 84.8%, with Glycine max
and Ricinus communis KASⅠ proteins, respecttively.
Moreover, phylogenetic analysis showed that AhKASⅠ
gene clustered with those from higher plants, and the
genes from cyanobacteria may be the origin of genes
from higher plants, mosses and eukaryotic algae. Real-
time PCR analysis revealed that the expression level of
AhKASⅠ was higher in leaf and seed than those in other
tissues. In addition, AhKASⅠ RNA was found in high
abundance at 45 and 65 DAP during seed development.
Plant seed oil production can be manipulated through
modifying the plant type Ⅱ FAS components. Despite
recent progress in detailed characterization of many
enzymes involved in plant fatty acid synthesis, the
mechanism of plant fatty acid synthesis is not well
understood (Ohlrogge and Jaworski, 1997). To increase
the expression of a single gene in a complex fatty acid
synthesis pathway is not effective in changing the
final product, unless concomitant changes in other en-
zymes involved are achieved. Therefore the regulation
should be spread out and coordinated among the many
enzymes involved in the pathway (Dehesh et al.,
2001). In the present study, the β-ketoacyl-ACP syn-
thase Ⅰ gene from peanut was identified. This enzyme
is critical to the elongation step and plays a pivotal
role in the regulation of the entire pathway (Magnuson
et al., 1993). This work may serve as a foundation for
further studies on the mechanisms regulating the ex-
pression of KASⅠ gene and provide candidate genes
for modifying oil quality via transgenic plants.
3 Materials and Methods
3.1 Plant materials
Peanut seeds (Arachis hypogaea
were sown in sand an
growth chamber under a 16 h~8 h light-dark cycle at
26℃and 22 ℃, respectively. Three kinds of 12-day-old
tissues including root, stem and leaves were collected
as experimental materials for quantitative real-time
RT-PCR analysis. In addition, the immature peanut seeds
from 25 days to 60 days after pegging (DAP) were
also collected for expression analysis. The peanut cul-
tivar was provided by Shandong Peanut Research In-
stitute, Qingdao, China.
3.2 Nucleic acid manipulation
Total RNA was extract
RNeasy Mini Kit (Qiagen) according
facturer’s instructions. The RNA samples were used
for real-time RT-PCR after RQ1 RNase-free DNase Ⅰ
(Promega, Wisconsin, USA) treatment to remove genomic