Page 5 - Legume Genomics and Genetics

Legume Genomics and Genetics (online), 2011, Vol. 2, No.3, 14-21
http://lgg.sophiapublisher.com
15
stress shock elements). In addition, HSFs as a kind of
stress signal response transcription factors, also
showed some resistance to cold and drought. There
are some evidences that plant HSFs have broadly
cross-protections under the different stress conditions
(Lee et al., 1995; Lee and Schöffl, 1996; Busch et al.,
2005).
In this study, we ligated the hsf8 gene into
pCAMBIA3300, a dicotyledonous plant expression
vector, by using soybean cotyledonary-node as explant,
the hsf8 gene was introduced into two novel lines,
Hajiao5337 and Hajiao5489, by Agrobacterium-med-
iated transformation. Real-time PCR method was
employed to determine the T1 transgenic plants for
obtaining the hsf8 gene highly expressing in
transgenic plants. The objectives of this study were to
achieve and improve expression levels of some
soybean target genes such as HSP70 through
expressing heat shock transcription factor hsf8 , to
enhance soybean tolerance to high temperature stress,
and to provide novel germplasm to develop
stress-resistant soybean varieties.
1 Results and Analysis
1.1 Construction of hsf8 gene expression vector
Plant gene expression vector was constructed
following technical scheme shown in Figure 1.
Plasmid pBI121-HSF8 was digested with EcoR Ⅰ
and Hind Ⅲ to be recovered in the size of
approximate 2.8 kb fragment (Figure 2, lane 1), which
contained the completely 35S + hsf8 + NOS
expression structure. Plasmid pCAMBIA3300 also
digested with EcoR Ⅰ and Hind Ⅲ restriction
enzymes, about 8.5 kb fragment was recovered
(Figure 2, lane 2), and then ligated both recovered
fragments. The ligated products were transformed into
E. coli DH5α. The recombinants were screened with
100 mg/L Kanamycin. The candidate recombinants
were validated by digestion of EcoR Ⅰ and Hind Ⅲ
restriction enzyme (Figure 2, lane 3), which can
regenerate the same size of pBI121-HSF8 and
pCAMBIA3300, named pCAMBIA3300-HSF8.
1.2 Selective pressure of glufosinate-ammonium on
soybean explants
According to preliminary results, concentrations of
glufosinate-ammonium were set up as 0 mg/L, 0.5
mg/L, 1 mg/L, 1.5 mg/L, 2 mg/L, 2.5 mg/L, 3 mg/L,
3.5 mg/L and 4 mg/L. in order to study the effects of
glufosinate-ammonium on the budding rate of explants
(Figure 3). The results showed the differential rate of
cotyledonary-node of Hajiao5337 and Hajiao5489 was
6.7% that was significantly less than that of the
control, but both of the differential buds showed
albino. Finally, 3.5 mg/L concentration
Figure 1 The scheme for constructing transformation vector
containing hsf8 gene
Figure 2 Plant expression vector pCAMBIA3300-HSF8
validated with endonucleases
Note: M: DL15000; 1: pBI121-HSF8 digested with EcoRⅠ
and HindⅢ ; 2: pCAMBIA3300 digested with EcoR Ⅰ and
HindⅢ; 3: pCAMBIA3300-HSF8 digested with EcoRⅠ and
HindⅢ