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Legume Genomics and Genetics (online), 2011, Vol. 2, No.3, 14-21
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16
of glufosinate-ammonium were determined as
criterion of selection pressure.
Figure 3 Effect of glufosinate-ammonium on the clustering
shoot regeneration of soybean cotyledonary-nodes of
Hajiao5337 and Hajiao5489
1.3 Target gene of T
1
generation plant validated
Plasmid pCAMBIA3300-HSF8 was used as a positive
control, non-transgenic regenerated plants and
Hajiao5337 and Hajiao5489 as references, sterile
water instead of template DNA as a negative control,
the total DNA of the resistant plants were used as
DNA template to amplify specific
bar
gene by using
35s-bar of primers. The results shown in Figure 4,
indicated that 17 resistant plants were exhibited
positive, which preliminary proved
hsf8
gene to be
integrated into the soybean genome.
Figure 4 The PCR analysis of genomic DNA in putative
transgenic soybean plants T
1
generation
Note: M: DL2000; 1: Positive control of pCAMBIA3300-HSF8;
2: Negative control of distill deionized water; 3~13: DNA of T
1
generation from independently transformed plants; 14: DNA
from untransformed plant Hajiao5337 (negative control); 15:
DNA from untransformed plant Hajiao5489 (negative control)
1.4
hsf8
expression level of T
1
generation plant
detected by Real-time PCR
The expression of
hsf8
gene in T
1
generation of
transgenic plants derived from Hajiao5337 and
Hajiao5489 as receptor were detected by Real-time
PCR, data shown in Table 1 and Table 2.
△△
CT
standed for different CT value between the target gene
hsf8
and reference gene (
lectin
). The expression level
of
hsf8
gene among tested plants were compared
based on differential expression in multiples (2
-
△△
CT
),
that is the ratio come from initial concentration of
hsf8
gene in transgenic plants to that of non-transgenic
plants. Nine individuals were exhibited significant
increases of gene expression, three of them were
increased twenty times than that of non-transgenic
plants, three plants for fifteen times increase and two
plants for five times increase (Figure 5). Compared
with the expression of Hajiao5489, the individual, T07
Ⅱ
-002-2, exhibited obvious differences in gene
expression level that was 48 times less than the
amount of gene expression of Hajiao 5489 (Figure 6).
Through the above results, the preliminary results
indicated that
hsf8
gene expression variations in
transgenic plants should be due to insertion of the
target gene
hsf8
.
2 Discussions
Soybean genetic transformation was considered as one
of the most difficult conundrums in soybean research,
to date soybean transformation system is still
inefficient and poor reproducibility. Increasing
transformation efficiency depends on the establishment
of perfect regeneration system, genetic transformation
system and smart screening strategy (Li et al., 2006;
Paz et al., 2006). In this paper, we adopted a modified
screening method, which recovery culture was carried
out prior to co-culture procedure. The results we
obtained were the similar with the previously reported
publication (Liu et al., 2007). It is noteworthy that
recovery culture time might be too long to inhibit the
growth of transformed cells due to excessive
proliferation and differentiation of non-transformed
cells, which may be inconductived to transformation
efficiency.
Bar
gene as a selective marker gene is less
restricted to receptor’s genotypes, which could be
used for transformation in almost all species, in this
point it would be helpful to improve the genetic
transformation efficiency.