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Legume Genomics and Genetics (online), 2011, Vol. 2, No.3, 14-21
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19
bacterial strain.
Northern blot method and RT-PCR quantitative
method are commonly used in gene expression
research. However, the Real-time PCR would be more
convenient, faster and more accurate than that of
Northern blot and RT-PCR to detect the abundance of
gene expression in a variety of tissue cells, to analyze
gene expression and regulation, to monitor mRNA
expression mode, to detect the presence of genes in a
small amount of tissue, to track the clone in the cell
population and to quantitatively analyze gene
transcription levels in different tissues (Livak and
Schmittgen, 2001).
In this study, we conducted
hsf8
expression analysis of
transgenic plants and non-transgenic plants by using
real-time PCR. Because introduced gene come from
soybean self, the non-transgenic soybean lines,
Hajiao5337 and hajiao5489, exhibited a reasonable
level of gene expression. In this research, there are
evidences of over-expression and low expression in
transgenic plants, the reasons might be increase of
gene copy numbers to enhance the level of expression,
whereas gene silencing might be the reason for low
expression in some transgenic plants.
3 Materials and Methods
3.1 Plant materials
Two high yield soybean new lines, Hajiao5337 and
Hajiao5439, developed by our research group, used to
be transformation receptors in this research.
3.2 Strains and plasmids
Plasmid pBI121-HSF8 containing
hsf8
gene and plant
expression vector pCAMBIA3300 were provided by
Dr Baoge Zhu from IGDB of CAS.
E. coli
strain DH 5
was bought from TIANGEN Company and
Agrobacterium tumefaciens
strains GV3101 was
deposited in this lab.
3.3 Chemicals and reagents
Taq
DNA Polymerase and T
4
DNA ligase were
purchased from TaKa-Ra Company; restriction
enzymes from Promega Corporation; Glufosinate-am-
monium, 6-Benzylaminopurine (6-BA) and Indole-3-u-
tyric (IBA) from Sigma company. RNAprep Plant Kit
from TIANGEN Company, Power SYBR® Green
PCR Master Mix and MicroAmpTM Optical Adhesive
Film Kit were purchased from ABI Research company,
and reverse transcription reagents were purchased
from Invitrogen. And sequencing primers were
synthesized by Shanghai Sangon. Antibiotics,
kanamycin and cephalosporin, were domestically
produced with analytical grade in China.
3.4
hsf8
expression vector construction based
pCAMBIA3300
Single colony, pBI121-HSF8 or pCAMBIA3300 was
picked to be cultured at 200 r/min shaking incubation
in LB liquid medium (Kan
+
) at 37
℃
until the OD
600
value equal 0. 4 measured. Plasmid pBI121-HSF8 was
digested with
Eco
R
Ⅰ
and
Hin
d
Ⅲ
restriction
enzyme, then about 2.8 kb fragments were recovered,
which contained the completely 35S +
hsf8
+ NOS
expression structure. While plasmid pCAMBIA3300
was digested with
Eco
R
Ⅰ
and
Hin
d
Ⅲ
restriction
enzyme, then about 8.5 kb fragments were recovered.
Two recovered fragments were ligated by T
4
ligase
overnight at 22
℃
. The connected products were
transformed into
E. coli
DH5 competent cells by heat
shock method. Positive recombinants were screened
by using kanamycin (Km) 100 mg/L, then positive
plasmids were transformed into
Agrobacterium
competent cells, and coated on plate of YEB (Kan
+
,
Rif
+
and Str
+
) for 48 h dark incubation until strain
plaque growing about 2 mm diameters.
3.5
Agrobacterium
mediated transformation
3.5.1 Screening concentration of glufosinate-amm-
onium
According to preliminary results, delay screening
method was employed in this research, The soybean
cotyledonary-node was applied to bud induction
culture medium without selecting agents about 3 days
until the differential bud spots on cotyledonary-node
appeared. Then the visible buds of cotyledonary-node
were transferred into bud induction media with
different concentrations of glufosinate-ammonium for
screening culture. Concentrations of glufosinate-amm-
onium were designed 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. Thirty explants were placed on media of each
concentration treatment with a duplicates and were
incubated for two weeks at (26±1)
℃
. The differential
rate of cotyledonary-node was calculated following as
the differential cotyledonary-node number by total
number of cotyledonary-node incubated.