Cloning Of crylA(b) Gene Into pCAMBIA 1301

Genetics Society of Malaysia
Cloning Of crylA(b) Gene Into pCAMBIA 1301
Norwati, A., and lRuslan, A.
Abslract
Cry or Bt genes isolated /iom Bacilhrs thuringiensis, encodes Jbr the production of
insecticidal crystal proteins, commonly known as "Cty" proteins. ClassiJicalion of
Cry: proteiru was based on insect specificity and nucleotide sequences. Of the three
Cry t A genes, [cry I A ( a), cryt I A(b) and cry I A ( c)J, products of ay I A ( b) were found
active against Lepidopteran and Dipteran larvae, making it a suitable candidate
lbr the developmenr of insect resistant plant through genetic engineering. The list
rtJ successful genetic tansformation oJ'monocots mediated by Agrobacterium
rumeiaciens has been increasing. Onefactor that contributes to the.se successes was
pCAMBIA I i0 I . Cassettes of gene of interest comprising 4 promoter, gene cocling
sequence and a terminator can be cloned into pCAMBIA 1301. The new recombinant p CAMB IA I j 0 I plasmid could t hen be us ed for transfonnation studies of bot h
dicots and monocot, either through Agrobacterium or particle bombardment. For
selection purposes, kanamycin and hygromycin could be used for bacteria (E.coli
and Agrobacterium) and planl, respectively. In this sudy crylA(b) gene was successfully cloned into 7CAMBIA 1301. This was confirmed through restriction mepping and Southern hybritlization. The new recombinant plasmid would then be
used in transformation o.tsessrnents Jbr teak improvement.
Introduction
Bacillus thuringiensis (Bt) is an insecticidal
bacterium. Bt is marketed worldwide for con-
tol
of many important plant pests, mainly Cat-
erpillars of the Lepidoptera (butterflies and
moths). Bt is also used for control of mosquito
larvae and stimuliid blackflies, a vector for river
blindness in Africa. Bt was first discovered in
.Iapan in 1902 in a silkworm-rearing unit. In
1911, it was isolated in a population of flour
moth and characterizedby Berliner in Thuringen
(Germany). Bacillus thuringiensis is a grampositive bacterium that synthesizes insecticidal
crystalline inclusions during sporulation. The
crystalline structure ofthe inclusion is made up
of protoxin subunits, called d-endotoxins. Most
B. thuingiensis strains produce several crystalline proteins (Cry proteins), each of which
shows a rather narow host range (Feitelson et.
al.,1992).
pCAMBIA
Forest Research Institute Malaysia (FRIM), Kepong;
rDept ofGenetics, Faculty ofLife Sciences, UKM Bangi
Emai I : norwati@frim. gov.my
2s8
I 30 1 is
becoming an increasingly
important vector in hansformation studies. It
was constructed with all the advantages of bi.
nary plasmid such as pIGl 12 I Hmbut with none
T'hird National Congress on Genetics, 1B-19 November l99B
of their drawbacks. The vector can be used for
routine cloning of gene cassettes of interest
a promoter, coding sequence of the
gene and terminator. The new constructs can
then be used for transformation of both dicots
and monocots either through Agrobacterium or
comprising
particle bombardment. The pUC18 multiple
cloning site (MCS) is proximal to the right border. pCAMBIA l30l hasgusA gene present
as a reporter, which can be used to assess the
efficiency of transformation. The multiple cloning site is positioned betweengusA (proximal
. The fragment was ligated into Hindl l l-digested and alkaline phosphatase-treated
pCAMBIA 1301. using T4 DNA ligase
(Maniatis et a|.,1982), and subsequently transformed into E. coll DH5a using the calcium
chloride method (Mandel and Higa, 1970).
White colonies containing putative recombinant
plasmids were selected on plates in the presence of isopropyl-b-D-thiogalactopyranoside
(IPTG) and 5-bromo-4-chloro-3-indolyl-b-D-
galactopyranoside (X-gal) and kanamycin
(Glonenborn et al., 197 8).
to the right border of T-DNA transfer) and
hygromycin (proximal to the left border). Thus,
translbmedplants expressing GUS activity and
Restriction mapping and hybridization
showing hygromycin resistance will also have
a cloned gene.
Mapping of restriction sites was performed by
Ligation of crylA(b.)/Bt gere into
pCAMBIA
1301 was attempted in effort to develop transformation techniques to be used in
teak improvement programmes. One of the
objective was to develop transgenic teak resistant to insect.
Materials And Methods
digestion of putative recombinant plasmid with
one restriction endonuclease, electrophoresed
lbr two hours in an 0.8% (wt/vol.) agarose gel.
The DNA was transferred onto a nylon membrane (Hybond N; Amersham) and was probed
against a Bt gene fragment labeled with the DIG
labeling kit (Boehringer Mannheim, 1995).
Similarly, Southern blouing (Southern, 1975)
was performed accordingly.
Bacterid strain and plasmid
pSB plasmid was used as source for the
cn,l A(b)/Bt gene with Es cherichia coli DH5a
as host. Whilst, pCAMBIA 1301 was used as
the target plasmid to receive the insert. E. coli
DH5a was routinely cultured in either liquid or
solid L broth ( l% trlrptone, 0.5olo yeast extract,
1% NACI). E. coli containing the pSB plasmid
was maintained in L broth supplemented with
50 ug/ml ampicillin. Bacteria containing the
putative recombinant derivatives of the plasmid
were maintained in L broth supplemented with
kanamycin ( l00ug/ml).
Isolation and cloning of crylA(b) gene
pSB plasmid was isolated using the alkaline lysis method (Bimboim and Doly, 1979). pSB
was digested with Hindl l l to yield a 2.915 kb
fragment which was subsequently isolated from
the agarose gel using the gene clean kit method
Results
crylA(b) gene and vector
As expected the Hindl I I -digested pSB plasmid
gave rise totwo fragments of 2.746kb and 2.915
kb in size. Based on the restriction map provided, the 2.915 kb fragment was expected to
contain the uylA(b) gene, the 35S promoter
and the NOS terminator. Whilst pGEM -42 vector was present in the 2.7 46 kb fragment. Similarly, Hindl I l-digested pCAMBIA 1301 gave
a linear fragment of about 11.837 kb (Figure
l).
Transformation
intoE coliDESa
Figure 2 shows the presence of two types of
colonies; white and blue. Selection and screening was done to confirm the stability of the recombinant plasmid. Plasmid extraction from the
selected colonies showed difference in size (Fig-
2s9
Genetics Society of Malaltsia
ure -1). Plasmids bigger than pCAMBIA 1301
were subsequently digested with two different
restriction enzymes to further confirm the presence of the inserts (Figure 4).
Restriction mapping and hybridizationPlasmids from two positive transformant
colonies were digested with different restriction enzymes (Figure 5 and 6). DNA was then
transfered onto nylon membrane and hybridrzed against the probe prepared earlier (Figure
7\.
Discussion
also hybridized to the vector
pCAMBIAl30l
due to the presence of the 35S promoter present
in pCAMBIAl30l, but there was no band for
marker Lambda/Hindlll (lane 1). Since the
probe hybridized to the blotted plasmid, thus this
confirmed the presence of the cry I A (b) gene in
the recombinant plasmid.
Acknowledgement
The work is supported by IRPA Grant 09-0401-0081. We would like to thank Ms Sharifah
Talib, Mohd. Rashdan Muad, and Ms Yuen for
their help.
Theoretically, when a colony is white, the lac z
gene was disturbed due to the presence of an
insert. However, restrictron digest carried out
on plasmid extracted from seven white colonres showed different sizes. Three colonies have
plasmid with the same size as pCAMBIAl301.
Two colonies contained plasmids smaller than
pCAMBIAl301. Whilst only two colonies
showed the preserce of plasmids bigger than
pCAMBIA.I 30 l. When the latter plasmids were
Birnboim, H.C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening
recombinant plasmid DNA. Nzcleic Acirls
Res.7: 1513-1523.
digested vvith Hind I I 1, it gave rise to an -2.915
and an -1 1.837 kb fragments, thus confirming
Dandekar, A. M., G. H. McGranahan, P. V. Vail,
S. L. Uratsu, C. A. Leslie, and J. S. Tebbets.
the presence of the insert within the vector
pCAMBIAl301 (Figure 4). The recombinant
plasmids were subsequently named pCAMBtl
andpCAMBt2.
Restriction mapping was performed to know
the orientation of the insert in both plasmids.
BamHl, EcoRl, EcoRV, Hindl l l, Pstl, Sacl,
Sall and Smal were used to digest the plasmid. Figure 5 and 6 shows differentbands than
those observed in BamHl (lane 5) and Sacl
(lane 10) digestions. pCAMBtl digested with
BamHl showed two bands about 2.1 kb and
12.6 kb while pCAMBt2 also showed two
bands, but the size were about 0.8 kb and I 1.8
kb. For Sacl digestion, pCAMBtl gave two
bands and the sizes were about 14.0 kb and 0.8
kb, pCAMBt2 also gave two bands about 12.6
kb. Thus, the orientation of both
plasmid are known (Figure 8).
kb and 2.2
Results for Southern blot of pCAMBtl are
shown in Figure 7. As mentioned earlier the
insert fragment was used as probe. The probe
260
References
Boehringer Mannheim, 1995. The DIG system
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Feitelson. J. S., J. Payne, L. Kim. 1992. Bacil-
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Kleiner, K. W., D. D. Ellis, B. H. McCown, and
K. F. Raffa. 1995. Field evaluation of
transgenic poplar expressing a Baci.llus
thuringiensis crylA(a) d-endotoxin gene
against forest tent caterpillar (Lepidoptera:
Lasiocampidae) and gypsy moth (Lepidoptera: Lymantriidae) following winter
dormancy. Environ. Entomol. 24(5) : I358I 364.
Mandel, M., and A. Higa. 1970. Calcium-dependent bacteriophage DNA infection. -i.
Mol. Biol. 53: 159-162.
Maniatis, T., E. F. Fritsch, and J. Sambrook.
1982. Molecular cloning : a laboratory
Third National Congress on Genetics, lB-t9 November l99B
manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
Southern, E.
M. 1975. Detection of specific
sequences among DNA fragments separated
by gel electophoresis..-I. Mol' Biol' 98" 5035
17.
kanamycin
Fig. 2. Two tlpes of transformant colonies on LB plate with 100 ug/ml
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Genetics Society of Malaysia
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Fig.l.
Insert (lane 5) and vector (lane 4)
before ligation
r234567E9101112
Fig.4. Plasmid digested with HindIII and PstI
t234567E910ilU13
Fig.6. pCAMBtl digested with different RE
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II
9 I0
ll
12 13 14 15 16 l7
Fig.3. Plasmid of different transformed
colonies
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Fig.S. pCAMBII2 digested with different RE
l312tll09 8 7 6 5 4 3 21
Fig.7. Representattive southern blot hybridization with the insert probe for the
pCAMBtl digestion
1998
Third National Congress on Genetics, tB-19 November
Hindi I I
35S promoter
cryl A(b)
35S prouroter
CANIV35S
crylA(b) gene
355 prolnoter
Hindl l l
Insert containing crylA(b) gene
Vector
Figure 8: Map for plasmid
pCAMBtl
pCAMBIAl30l
and pCAMBt2
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