Page 5 - Maize Genomics and Genetics

Maize Genomics and Genetics 2012, Vol.3, No.1, 1- 5
ISSN 1925-1971 http://mgg.sophiapublisher.com
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In this paper, we attempted to construct GBSS Ⅰ
(granule bound starch synthaseⅠ ), a key gene of
wheat amylose synthesis, and HorD, a specific
promoter for barley endosperm, into highly efficient
plant expression vector. By over expression of wheat
GBSS Ⅰgene, in specific stages, we found that this
gene expressed massively in maize endosperm. And
we used the somatic embryo subcultured around 20 d
as the receptor to obtain a wheat GBSS Ⅰtransgenic
maize plants successfully by Agrobacterium mediated
genetic transformation methods (Li, 2011).
1 Results and Analysis
1.1 Cloning of wheat GBSS Ⅰgene and barley
endosperm specific promoter HorD gene
In this experiment, we designed the specific primers
based on the wheat GBSS Ⅰgene sequence, and used
wheat genomic DNA as template for PCR amplification.
Then the recycling target bands were connected with
pGM-T vector, which was transformed into E. coli
DH5α, and the recombinant plasmid was sequenced.
Comparison of sequencing results showed that the full
length of the wheat GBSS Ⅰgene was 1 853 bp in
size (Figure 1A); Based on the barley endosperm
specific promoter HorD, we designed the specific
primers. And barley genomic DNA was used as
template for PCR amplification. Recycling target
bands were obtained and connected to pGM-T vector,
And then the vector was transformed into E. coli
DH5α and sequenced. Compared to the sequence, the
results showed that the full length of promoter HorD
was 510 bp in size (Figure 1B).
1.2 Construction of the plant expression vector
The plant expression vector was constructed successfully.
Figure 1 GBSS Ⅰand HorD amplified from common wheat
and barley, respectively
Note: M: DL2000 marker; 1: GBSS ⅠPCR amplification; 2:
HorD PCR amplification
And the result was validated by PCR and restriction
enzyme digestion, and it was consistent with the expected
result in size, which was 2 300 bp in size (Figure 2;
Figure 3), which suggested that the vector pHorD-
GBSS Ⅰbe consistent with our experimental design.
Figure 2 Plant expression vector pHorD-GBSSⅠ was identified
by PCR
Note: M: DL2000 marker; 2: The PCR fragment of plant
expression vector pHorD-GBSS Ⅰ
Figure 3 Plant expression vector pHorD-GBSS Ⅰ was
identified by double enzymic digestion
Note: M: λ-HindⅢ marker; 2: The digested fragment of plant
expression vector pHorD- GBSS Ⅰ
1.3 Agrobacterium mediated genetic transformation
of maize
Somatic cell embryos subculture of about 20 d was
transformed by Agrobacterium mediated method. The
callus were cultivated in co-culture medium and in
dark for 3 d at 25℃ after infection. Then they were
transferred into bacteriostatic subculture medium for
10 d, and then were transferred into the inhibitory
differentiation medium for differentiation. The results
displayed that 113 regeneration plants were obtained
after 45 d (Figure 4).
1.4 Molecular detection of transgenic plant
Total DNA was extracted from the leaves of regene-
ration plant by CTAB method. Firstly, bar gene
primers, which were designed previously, were used
for PCR detection (Figure 5). There were 5 plants can