Page 6 - BM 2011 Vol.2 No.3

Bioscience Methods
BM 2011, Vol.2, No.3 http://bm.sophiapublisher.com
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for the constructs. A 3.5 kb target band for pBI121-
Cry1Ac22F (Figure 5), and a 1.9 kb target band for
pBI121-Cry1Ac22T (Figure 6), were amplified that
indicated both of them have been transformed into
Agrobacterium EHA105 successfully.
Figure 5 PCR identification of pB1121-Cry1Ac22F
Note: M: λ DNA/HindⅢ; 1~10: Positive transformants
Figure 6 PCR identification of pB1121-Cry1Ac22T
Note: M: λ DNA/HindⅢ; 1~10: Positive transformants
1.4 Identification of of transgenic Arabidopsis thaliana
The cry1Ac22 full length gene and cry1Ac22 truncated
gene were transformed into the genome of Arabidopsis
thaliana plants mediated by Agrobacterium, respectively.
We identified that the plants of No.3, No.4, No.6,
No.7, No.8 and No.10 were exsiting the 3.5 kb
cry1Ac22 full length fragment, (Figure 7).
Whereas for truncated cry1Ac22T transformation, we
found that cry1Ac22T gene has been integrated into
Arabidopsis thaliana plants with the plants of No.3,
No.5, No.6, No.9, No.10 (Figure 8).
Figure 7 Identification of transgenic cry1Ac22F plants
Note: M: DL2000 plus ladder, 1: Negative control (DNA from
non-transgenic plants); 2: Positive control (pBI121- Cry1Ac22);
3~10: DNA from putative transgenic plants
Figure 8 Identification of transgenic cry1Ac22T plants
Note: M: DL2000 plus ladder, 1: Negative control (DNA from
non-transgenic plants); 2: Positive control (pBI121-Cry1Ac22T);
3~10: DNA from putative transgenic plants
2 Discussions
Bacillus thuringiensis cry1A toxin genes are the most
commonly used as insecticidal targeted gene, among
them, cry1Ac gene was extensively used for developing
transgenic Bt cotton. There are numbers of reports that
the full length cry1A genes were transformed into
many crops including cotton, rice, potato, corn, canola,
soybean, sugarcane, peanut etc. but the amounts of Bt
toxins expressed in these transgenic plants were
considerable low and were not enough toxic dose to
the targeted pests (Romeis et al., 2006). These researches
indicated that the full length sequence of the cry1A
gene be not suitable for developing transgenic crops.
Therefore, enhancement of Bt toxicity in transgenic
plants would be essential goals in Bt transgenic breeding.
In this research, we designed two kinds of plant
expression constructs, pBI121-Cry1Ac22F and pBI1
21-Cry1Ac22T, and transformed into the model of
dicot plant, Arabidopsis thaliana. Both of the gene were
transformed into Arabidopsis genome. The preliminary
studies on the expressed toxin in Arabidopsis plant
indicated that the expression of truncated domain be
higher than that of full length gene (data not showed).
We believed that the Bt toxin shoud be well designed
for transgenic plants than completely employed from
those deposited in GenBank.
3 Materials and Methods
3.1 Materials and reagent
Escherichia coli JM109, pMD18-T vector, pBI121
plant expression vector were deposited in our lab.
DNA restriction endonucleases Sac Ⅰ, BamHⅠ , DNA
Marker, T4 DNA ligase, Taq DNA polymerase and