Page 5 - BM 2011 Vol.2 No.3

Bioscience Methods
BM 2011, Vol.2, No.3 http://bm.sophiapublisher.com
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hydrolysed to the 65 kD core protein (Lenin et al.,
2001). Therefore, it is essential to carry out the
research to express the total length cry1Ac22 and the
functional domain from Bt W015-1.
In the experiment, we cloned the full length of cry1Ac22
gene of 3 534 bp in length and its function domains of
1 959 bp by PCR, and constructed them to plant
expression vector pBI121 to make the constructs of
pBI121-Cry1Ac22F and pBI121- Cry1Ac22T both
carrying with Kanamycin selection marker.
Arabidopsis thaliana was transformed during the flowering
stage mediated by Agrobacterium tumufaciens, lots of
transgenic Arabidopsis thaliana seeds with positive
Kanamycin resistance were harvested in this research
that facilitate the understanding of Bt toxin functions
in plant transgenic breeding.
1 Results
1.1 Cloning of the full length cry1Ac22 gene and its
function domain
cry1Ac22 full length gene of 3 534 bp (Figure 1A) and
cry1Ac22 function domain of 1 959 bp (Figure 2A)
were amplified by specific primers. Both of the target
genes were ligated to pMD18-T vector and positive
clones were identified by enzymetic digestion of
BamH and
Ⅰ SalⅠ , respectively (Figure 1B). And
then named as pMD18-T-Cry1Ac22F for full length
gene (Figure 1C) and pMD18-T-Cry1Ac22T for
truncated gene (Figure 2B), respectively. Finally, we
sequenced the positive clones to validate the sequence
of Cry 1Ac22 by GenBank database.
1.2 Plant expression construct of pBI121-Cry1Ac22F
and pBI121-Cry1Ac22F
Full length gene cry1Ac22 cutting from recombinant
Figure 1 PCR amplification of cry1Ac22 and digestion of pMD18-
T-Cry1Ac22F by restriction enzymes
Note: M: λ DNA/HindⅢ; A: 1: cry1Ac22; B: 1: pMD18-T; C: 1:
Digestion of pMD18-T-Cry1Ac22F with BamH and
Ⅰ SalⅠ
Figure 2 PCR amplification of cry1Ac22T and digestion of
cry1Ac22T with BamH and
Ⅰ SalⅠ
Note: M: λ DNA/ Hind Ⅲ; A: 1: cry1Ac22T; B: 1: Digestion of
pMD18- T-Cry1Ac22T with BamH Ⅰand SalⅠ
plasmid pMD18-T-Cry1Ac22 was ligated into plant
expression vector pBI121 to make a plant expression
construct named as pBI121 -Cry1Ac22F, and then was
transformed into Escherichia coli JM109. While the
construct of pBI121-Cry1Ac22T for truncated gene
was constructed by the same way. Both of the
constructs were validated by digesting with Bam HⅠ
and Sal Ⅰ. A 3.5 kb target fragment and a 18 kb vector
fragment was cut for pBI121-Cry1Ac22F (Figure 3).
Whereas a 1.9 kb target fragment and a 18 kb vector
fragment for pBI121-Cry1Ac22T (Figure 4).
Figure 3 Digestion of pBI121- Cry1Ac22F with BamH andⅠ SalⅠ
Note: M: λ DNA/Hind Ⅲ ; 1~6: Transformants; 1,2,4,5,6:
Positive clones
Figure 4 Digestion of pBI121- Cry1Ac22T with BamH andⅠ SalⅠ
Note: M: λ DNA/Hind Ⅲ ; 1~6: Transformants; 1,3,4,5,6:
Positive clones
1.3 Identification of Agrobacterium recombinant
plasmid by PCR
The constructs were transformed into Agrobacterium
EHA105, and the plasmids were extracted by using
commercial extract kits. The positive clones were
amplified and identified by the same specific primers as