clontng of a cryiiia endotoxin gen bt

7/28/2019 Clontng of a CryIIIA Endotoxin Gen Bt

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J. Biosci., Vol. 21, Number 5, September 1996, pp 673685. Printed in India.

Cloning of acryIIIA endotoxin gene ofBacillusthuringiensis var.tenebrionis and its transient expression

in indica rice

TONY M JOHNSON*, A S RISHI**, PRITILATA NAYAK andSOUMITRA K SENPlant Molecular and Cellular Genetics and Centre for Plant Molecular Biology, Bose

Institute, Centenary Building, P1/12, CIT Scheme VII-M, Calcutta 700 054, India

Present address: *Department of Biochemistry, College of Biological Sciences, 140 Gortner

Laboratory, 1479 Gortner Avenue, St. Paul, MN 55108-1022, USA

**Biotechnology Division, Rallis India Limited, Rallis Agrochemical Research Station,

Bangalore 560 058, India

MS received 1 September 1995; revised 13 June 1996

Abstract. Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus

oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is

quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was

considered desirable. CryIIIA endotoxin ofBacillus thuringiensis var.tenebrionis, a 65 kDa protein

toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was

isolated from a local isolate ofBacillus thuringiensis var.tenebrionis. The gene was tailored at the

N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon

next to ATG of threonine to proline. This modification did not affect the functional property of the

gene product. A chimeric construct of the modifiedcryIIIA gene was developed containing CaMV35S

promoter andnos terminator for plant expression. The expressibility of thecryIIIA gene in indica ricewas judged through test for transient expression in indica rice protoplasts.

Keywords. Bt cryIIIA gene; coleopteran rice pest; N-terminal truncation; sandwich ELISA;

transient gene expression.

1. Introduction

Postharvest loss due to storage grain pests in cereals and seed legumes is enormously high

in developing countries due to inadequate protection measures practised for economic

reasons against these pests. Researchers have focused on the possible ways to endow plantswith improved survival in field by generating transgenics with insecticidal crystal protein

(ICP) genes ofBacillus thuringiensis (Bt) (Delannay et al1989; Gasser and Fraley 1992;Wilson et al1992), proteinase inhibitor genes (Hilderet al1987; Johnson et al1989), genes

for lectins (Chrispeels and Raikhel 1991) and -amylase inhibitors (Huesing et al1991;Shade et al1994). It is known that rice weevil (Sitophilus oryzeae L.) is the most destructive

insect pest of grains in the world. Additionally, rice hispa (Dicladispaarmigera), another

coleopteran insect pest of growing rice plants causes considerable damage (Herdt 1991).

The strategy to develop insect resistant crop plants by makingBtgene express in plants has

already yielded useful results in certain crop plants including cotton, rape and corn. The

ICP produced byB. thuringiensis var. tenebrionis (Btt) is a 65 kDa -endotoxin of CryIIIA

Corresponding author (Fax, 091-33-343886; Email, [email protected]).

673


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674 Tony M Jonson et al

type (Hofte and Whitely 1989), toxic to coleopteran insects. A strain ofBttproducing crystal

protein of CryIIIA type was isolated locally. On feeding tests, the grubs of rice weevil were found

to be sensitive to the crystal protein produced by this isolate.

In pursuance of our interest in the ultimate development ofBttransgenic crop plants,we report in this communication our success in cloning a cryIIIA gene from the

bacterial strain that we isolated. The peptide expressed by the Bt gene in Escherichia

coli was found to be toxic to the larvae of rice weevil. Furthermore, it was observed that

the gene could transiently express in rice protoplasts.

2. Materials and methods

21 Isolation and characterization of the local isolate of Btt

A strain ofBttwas isolated from rice grain dust. The identification and characterizationof the strain was carried out on the basis of tests carried out at Kyoshu University,

Japan; and on the basis of crystal protein character, entomocidal property and

immunological analysis as revealed through ELISA using a reference protein of

CryIIIA (Krieg et al1983), as standard.

2.2 Purification of ICP from Btt

Crystal proteins were purified from the sporulated culture of the isolated strain ofBtt,

following the procedure of McPherson et al (1988). SDS-PAGE analysis revealed that

the purified CryIIIA protein contained a single polypeptide of 65 kDa (figure 1).Polyclonal antibodies were raised in rabbit against the 65 kDa protein and the

specificity of the polyclonal antibody was confirmed by immunoblot analysis.

23 Production of monoclonal antibodies

Five monoclonal antibodies (Mab) against the 65 kDa CryIIIA protein were produced

in the laboratory following essentially the method of Kohler and Milstein (1975).

Spleen cells from immunized mice and the non-immunoglobulin producing mouse

myeloma cells (P3 63.Ag8.653) were fused at the ratio of 3:1 using polyethylene glycol

(PEG, MW 1500, Boehringer Mannheim). Hybrids were selected in Dulbecco modified

eagles medium (Nissui Pharmaceutical Co. Ltd) containing hypoxanthine-aminop-

terine-thymidine (GIBCO-BRL). Antibody secreting hybrid cells were selected by

indirect enzyme-linked immunosorbent assay (ELISA). Cell lines producing antibodies

were cloned by limiting dilution (Harlow and Lane 1988), using peritoneal macrophage

from unimmunized Balb/c as feeder layer. Expanded cells were injected in-

traperitoneally with pristane (2,6,10,14-tetramethyl pentadecane) in Balb/c mice for

ascitis fluid. Monoclonal IgG was purified by ammonium sulphate (45%) precipitation

followed by DEAE-cellulose column chromatography (Harlow and Lane I988).

2.4 Indirect ELISA

Indirect ELISA was performed according to Harlow and Lane (1988). Culture super-

natants were used as primary antibody. Goat ant-mouse IgG + IgM + IgA alkaline


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Cloning of cryIIIA gene and its expression in rice 675

phosphate conjugate and p-nitrophenyl phosphate (p-NPP) were used as secondary

antibody and substrate, respectively. Optical density was measured at 450 nm in anELISA reader (Anthos).

2.5 Isolation of the cryIIIA gene

Based on the earlier report (Sekar et al 1987), we took a chance by assuming that the

cryIIIA gene in the local isolate is also contained in a 59 kb BamHI fragment of the

plasmid DNA. Thus, the total plasmid DNA of the Bttstrain was digested with BamHI

endonuclease and the ~ 59 kb fragments were shotgun cloned into pUC18 and

transformed into E. coli strain DH5. Bacterial colonies containing recombinant

plasmids were screened through antigen-antibody assay system against the toxin for

the presence of the cryIIIA gene through colony hybridization using rabbit polyclonal

antibodies as probe. Fortunately, one positive clone (pBTT1) could be isolated(figure 2). The cloned BamHI fragment revealed that the restriction cleavage pattern

(figure 3) of theBamHI fragment and its partial nucleotide sequences were similar to theearlier known cryIIIA gene (Sekaret al1987; McPherson et al1988).

The 59 kb BamHI cleaved fragment containing the cryIIIA gene cloned in pUC18

(pBTT1) produced two bands of 72 kDa and 65 kDa (figure 4) in E. coli. The twoproteins were generated quite likely through the help of the Bt promoter already

contained in the cloned fragment and the two ATG codons present in the same open

reading frame of the cryIIIA gene (McPherson et al1988). It has already been reported

Figures 13. (1) SDS-PAGE analysis of the crystal protein purified from the local isolate of

Bacillus thuringiensis var. tenebrionis. Lane 1, molecular weight markers in kDa and lane 2, the Cry

protein (4 g) purified from the local isolate. (2) Colony hybridization using rabbit polyclonalantibody as probe for the identification of the clone (arrow) containing the ICP gene. ( 3) Agarose

gel electrophorogram of pBTT1 DNA cleaved with restriction endonucleases: lane 1,BamHI; lane

2,HindIII; lane 3,PstI, lane 4,EcoRI cleaved pBTT2; lane 5,HindIII cleaved -DNA.


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Figure 4. Immunoblot analysis: lane 1, the purified CryIIIA protein from B. thuringiensis var

tenebrionis; lane 2, purified protein fromE. coli cells containing the cloned cryIIIA gene.

Figure 5. (A) Strategy adopted for truncation of the N-terminal end of the cryIIIA gene. (B,

BamHI, Bg, BglII, E, EcoRI, H, HindIII, P, PstI, Sp, SphI). (B) Details of N-terminal

modification with the sequences involved.


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that the Bt promoters can express in E. coli albeit poorly (Adang et al 1985), which

however, can be immunologically detected. Both the polypeptides are known to beequally toxic to insects (McPherson et al1988).

In order to facilitate plant expression of the cryIIIA gene, the following experimentalapproach was followed (figure 5A) to truncate the N-terminal end of the cryIIIA gene

near the second ATG codon. A 27 kb Pstl fragment of the ICP gene contained in

pBTT1 was isolated and cloned into pUC18 at the PstI site to generate pBTT2. The

PstI site is situated 6 nucleotides downstream to the second ATG codon of the gene

(figure 5A, B). The cloning of the cryIIIA gene took advantage of the ATG sequences of

the SphI site in the multiple cloning sites of pUC18 as shown in figure 5B. This in

effect caused replacement of one codon for threonine to proline, next to ATG. Proper

orientation of the ligation product was ascertained through restriction with HindIII.

2.6 Expression of the cryIIIA gene in E. coli

Expression of the cloned cryIIIA gene was tested inE. coli by utilising pET-3, a bacterial

expression vector (Studieret al1990). The pET-3 is a transcriptional expression vector

containing a T7 promoter situated between 23 to + 26 followed by the unique BamHI

cloning site with no potential translational initiation site. It also has a transcriptional

terminator Tdownstream toBamHI site. AHindIII cleaved ~ 23 kb fragment of the gene

derived from pBTT2 was cloned into BamHI site of pET-3. The cloning was carried out

after all the cut ends of the DNA were endfilled with Klenow, followed by blunt end

ligation. The recombinant DNA was transformed into E. coli strain BL-21 (DE3).Induction of the transformed colonies with 01 mM IPTG resulted in the synthesis of

T7 RNA polymerase which eventually transcribed the modified cryIIIA gene cloned underthe control of T7 promoter. Screening of the transformants was carried out through colonyhybridization using rabbit polyclonal antibodies. The transformants further showed that

the gene product contained normal immunological properties, as revealed when chal-

lenged individually with five different Mabs (figure 6).

Figure 6. Immunoblot analysis of the protein expressed in E. coli by the truncated cryIIIA

gene using 5 different Mabs raised against the 65 kDa CryIIIA protein (lane 1, Mab/Btt1; lane

2, Mab/Btt2; lane 3, Mab/Btt3; lane 4, Mab/Btt4; lane 5, Mab/Btt5).


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2.7 Detection of ICP expressed in E. coli

E. coli cells of strain BL-21 (DE3) were grown overnight in L-broth, pelleted and

concentrated by suspending in 10 mM NaCl, 10 mM Tris HCl (pH 80) and 1mMEDTA containing PMSF at 200 ng/ml. The suspension was sonicated on ice with three

30 s bursts at maximum power (Labsonic 2000, B. Braun) and the extracts were stored

at 20C. Protein concentration was determined following Bradford (1976) method.

Crystals and E. coli extracts were then solubilized in cracking buffer (1% SDS, 2%

2-mercaptoethanol, 6 M urea, 001 M sodium phosphate, pH 72) by heating at 95C for

5 min.

SDS-PAGE was carried out following the method of Laemmli (1970) and immunob-

lot analysis was done following the method of Towbin et al (1979) where goat-anti-

rabbit-HRP (GIBCO-BRL) was used as the secondary antibody.

2.8 Transient expression of the ICP gene in the rice protoplasts

28a PEG uptake of DNA into rice protoplasts: Embryogenic cell suspension cul-

tures of Oryzasativa sub sp. indica ev. Heera were used as the experimental material.

Three days after subculture, 1 g of the suspension cells were mixed with 10 ml of

enzyme mixture containing 2% cellulase Onozuka R10, 01% pectolyase Y23, CPW

salts (Fearson et al 1973), 04 M mannitol (pH 60) and incubated for 34 h in dark

at 28C. The isolated protoplasts were sieved through a 30 m nylon mesh, centrifuged

at 100gfor 5 min and the pellet was resuspended in 1 ml of CPW medium (Fearson et al

1973), floated on top of 5 ml of 20% sucrose and centrifuged for 10 min at 100 g.Protoplasts forming a ring on top of the sucrose layer were collected and washed

once with CPW medium. The viability of the isolated protoplasts were tested with

FDA staining. The density of the viable protoplasts was adjusted to 1 107 proto-

plasts/ml.

PEG (40%, MW 8000) was prepared by dissolving in Krens F medium (Krens et al

1982) without CaCl2.2H2 O. 2M CaCl22H2 O was prepared separately, autoclaved

and then added to the PEG solution to bring CaCl2 to a final concentration of 125 mM.

One ml of protoplast suspension was taken in a 50 ml conical bottom tube, 50 l of

pCNMBTT DNA (1 g/l) was added to the protoplasts. One ml of the PEG solution

was added dropwise to the protoplasts and mixed slowly, incubated at 45C for 5minfollowed by 20 s in ice and 25 min in 30C. Krens F medium was added dropwise

initially to the protoplast-PEG mixture and then 500 l to 1 ml fractions over a period

of 30 min to bring the total volume to 30 ml. The protoplasts were then centrifuged at

100 g for 15 min. The resultant pellet was washed with Kpr medium (modified Kao

medium, Lee et al 1989) once and resuspended in 1 ml of the same medium. The

viability of the protoplasts was tested and the final density of the viable protoplasts was

adjusted to 12 106/ml prior to plating.

2XKpr medium was mixed with an equal volume of autoclaved 16% SeePlaque

agarose in water in 45C. Six ml compact volume of suspension cells were suspended in

the above medium and poured into 70 mm petri-plates and allowed to solidify.

Membrane filters of 08 m pore size were laid on top of the agarose feeder layer.

Protoplast suspension (200 l) was spread on top of the membrane filter, the petri-

plates were sealed with parafilm and incubated in dark at 24C.


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2.8b Protein isolation: After 48 h of incubation, the protoplasts were salvaged from

the membranes and suspended in extraction buffer (1:1w/v) containing 50 mM Tris

(pH 95),100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 M leupeptin. The samples were

sonicated briefly and homogenized for 30 s. The materials were then incubated for 1 h at4C and the pH was equilibrated to 85 with 01 N HCl followed by centrifugation

(16,000 g) at 4C for 30 min. The supernatant was aspirated out and subjected to a 20%

(w/v) (NH4)2 SO4 precipitation followed by centrifugation (37,000 g) at 4C for 30 min.

The supernatant was collected and dialysed against 50 mM Tris buffer (pH 85)

containing 100 mM NaCl. The dialysed samples were affinity purified by mixing with

rabbit polyclonal antibody (anti-CryIIIA)-Sepharose (50 l/m1) and incubated over-

night at 4C. The material was then centrifuged (1,000g) and the agarose pellet was

treated with minimum volume of 200 mM Gly (pH 25), for 25 min followed by

centrifugation (1,000g) for 1 min and the suprnatant was immediately treated with

1/10 th volume of 1 M Tris buffer (pH 85). The samples were then advanced for ELISA.The treatment samples were utilized for tests after determining the soluble protein

concentration in each sample by the method of Bradford (1976) using BSA as the

protein standard.

2.8c Sandwich ELISA: Polystyrene microtitre plates were coated with rabbit poly-

clonal antibodies (1:300 dilution), resuspended in 10 mM PBS and kept overnight at

4C. The wells were blocked with 150l 1% BSA, incubated for 1 h at 37C and washed

with 10 mM PBS. Proteins extracted from the cultured rice protoplasts were added to

each well and incubated for 2 h at 37C. After washing away the unbound proteins,

single Mab/Btt2 (Johnson 1993) was added to each well and incubated for 1 h at 37C.

Goat anti-mouse (IgG + IgM + IgA) alkaline phosphate conjugate was added and thepresence of antigen was determined by using p-nitrophenyl phosphate as substrate

dissolved in diethanolamine buffer (pH 95). Detection limit of ELISA was determined

by making a standard curve using the CryIIIA protein. The concentration of the

antigen present from treatment samples, judged in 5 replications in each case of three

separate experimental attempts, was determined from the standard curve drawn from

the known antigen as control.

2.9 Insect bioassay

The rice weevils were reared in the laboratory for 34 generations on rice flour.

Laboratory grown first instar larvae were collected immediately after their eclosion.

They were then cultured in small tubes with provison for aeration in incubator at 28C

and 75% relative humidity. Each tube contained 10 larvae and 10 g of rice flour as food.

Each treatment lines for bioassay was represented by 3 replicates. The toxin expressed

inBtandE. coli were isolated separately as described previously except that PMSF was

excluded from the sonication buffer. Dehusked rice grains were powdered to homo-

geneity to which the toxin was added. The mixing was carried out in a manner so that

the food offered to the larvae contained 0 and 40 ng of the toxin peptide per mg of the

total soluble rice flour protein. The LC50 of ICP ofBttof a standard strain (Kreig et al

1983) was estimated to be ~ 48 ng/mg of the rice flour protein. The duration of each

bioassay was fixed for 1 month. Effects of the toxin on the survival and developmental

cycle of the feeding insect grubs were recorded (table 1).


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Table 1. Effect on insect survival and metamorphosis when

the insects were fed with 40 ng ICP/mg RFP*.

*RFP, Rice flour protein

3. Results

3.1 Cloning of the cryIIIA gene into plant expression vector

The isolated cryIIIA gene was cloned into a plant expression vector, pCN18 (figure 7A).

The pCN18 had been developed by transferring theEcoRI-HindIII fragment of the plant

vector pBI121 (Jefferson et al1987) into pUC18 to generate pCGN18 and the uidA gene

contained in this transferred EcoRI-HindIII fragment was eliminated through restriction

at the SmaI and SstI sites followed by blunt end ligation after end-filling with Klenow

(figure 7A). The cryIIIA gene contained in pBTT2 was then restricted with HindIII and

the ~ 23 kb fragment was blunt end ligated into the BamHI site of pCN18 after

end-filling with Klenow (figure 7B). We selected a clone (pCNBTT) where the cryIIIA

gene insert could be recovered by digestion with BamHI andBglII (figure 8A). That this

22 kb BamHI-BglII fragment contained the cryIIIA gene was ascertained by cloning it

into the bacterial expression vector pET-3 (pETB). Immuno blot analysis (figure 9)

confirmed that the 22 kb BamHI-BglII fragment contained the functional cryIIIA gene

which produced a 65 kDa protein. This 22 kb fragment was then cloned into theBamHI

site of pCNM18 (figure 7B), a modified form of pCN18. The modification was carried out

in the manner, as shown in figure 7A. A 10 kb PstI-EcoRI fragment containing the

CaMV35S promoter and the nos terminator from pCN18 was eluted and endfilled with

Klenow. Simultaneously, pUC18 was digested withPvuII, the 24 kb fragment was elutedfrom the gel and ligated with the endfilled 10 kb PstI-EcoRI fragment from pCN18

to create pCNM18. This vector contained BamHI and XbaI sites downstream to

CaMV35S promoter and an EcoRI site downstream to the nos terminator. The EcoRI

site could be recovered by endfilling with Klenow followed by ligation at PvuII site.

Subsequently, the 22 kb fragment of DNA generated from pCNBTT after digestion

with BglII + BamHI, was cloned into pCNM18 to generate pCNMBTT (figure 7B).

Restriction analysis of the cloned modified cryIIIA gene in pCNMBTT confirmed the

presence of the characteristic 07 kb EcoRI fragment of the cryIIIA gene. Orientation of

the modified cryIIIA gene with respect to CaMV35S promoter was checked by

digestion with BamHI-EcoRI (figure 8B). The right orientation of the cryIIIA gene tothe promoter was confirmed through the evidence for generation of 07, 085, 085 and

32 kb fragments, as in case of opposite orientation, production of 02, 07, 085 and

385 kb DNA fragments would have resulted.


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Figure 7. Cloning scheme for generation of (A) pCNM18 and (B) pCNMBTT (B,BamHI, Bg,

BglII, E, EcoRI, H, HindIII P, PstI, Pv, PvuII, S, SphI, Sm, SmaI, Ss, SstI, X, XbaI, Mcs,

multiple cloning site).


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Figures 8 and 9. (8) Agarose gel electrophoresis of pCNMBTTDNA cleaved with restriction

endonucleases. (A) Lane 1 and lane 3,EcoRI, lane 2,BamHI-BglII and lane 4,PstI. (B) Lane 1,

uncut DNA; lane 2,EcoRI cleaved DNA; lane 3, cleaved withBamHI-EcoRI showing 32, 085,

085 and 07 kb fragments; lane 4, HindIII cleaved -DNA. (9) Immunoblot analysis of theprotein purified from the cryIIIA gene contained in 22 kb BamHI-BglII fragment in pET-3

(lane 1, 65 kDa; lane 2, negative control).

3.2 Transient expression of the cryIIIA gene in rice protoplasts

Transient expression of the cryIIIA gene contained in pCNMBTT in rice was studied

by introducing the gene into viable rice protoplasts through PEG uptake. Polyclonal-monoclonal Sandwich ELISA was used for monitoring the CryIIIA protein in five

replicate experiments. It could be evidenced that the chimeric construct containing the

cryIIIA gene was capable of expressing in rice protoplasts. The levels of transient

expression after 48 h of incubtion of protoplasts ranged between 00008 to 0001% of itstotal soluble protein.

4. Discussion

Truncation of the N-terminal end of the cryIIIA gene was carried out in the present

study to clone a shorter form of the gene to facilitate its expression in plant (Rhim et al

1990). The cryIIIA gene, when expressed in E. coli directs the synthesis of a 72 kDa

protein. However, in nature the 72 kDa protein is further processed to a 65 kDa protein

by spore-associated bacterial proteases which remove the first 57 N-terminal amino

acids (McPherson et al1988). On the contrary, it is known that the 3'end of the genecan not be truncated, as it causes loss of the toxic activity (Hofte and Whiteley 1989). In

the present study, modification of the gene by deletion at the N-terminal end, addition

of a synthetic ATG codon and replacement of the codon, threonine to proline, has been


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seen not to alter the immunological as well as entomocidal property of the expressed

protein (table 1). A truncated form of the cryIIIA gene with a deletion at the 5'end has

shown not to affect protein function (Rhim et al1990).

Polyclonal-monoclonal antibody sandwich ELISA was adopted in the present study fordetecting the presence of the CryIIIA protein. The Mab/Btt2 was used for the purpose its

efficiency was similar to pooled Mabs, for detecting the CryIIIA protein in a mixture of

antigens (Johnson 1993). We could detect as low as 2 ng of the CryIIIA protein in a given

sample of protein. The two antibody sandwich ELISA is known to be fairly accurate and

sensitive (Harlow and Lane 1988). That use of monoclone based technique can be used for

detection and quantification of Cry protein has been demonstrated earlier (Vaeck et al

1987). The past workers could detect as low as 100 pg to 1 ng of the protein by the use of

a specific Mab. The transient expression by the rice protoplasts indicated that the chimeric

construct of the cryIIIA gene is expressive in rice. The levels of transient expression of the

native gene in the present case could be immunologically detected.Bacterial cry genes are known to be inefficient for expression in plant. The slow rate

and/or low level of nuclear production of full-length mRNA ofBtgene is an important

cause (Murray et al1991) for the relatively low expression levels of Cry proteins in plant

cells. TheBtgenes are excessively AT-rich as compared to normal plant genes. The bias

in nucleotide composition of the DNA could have a number of deleterious cones-

quences on gene expression, since AT-rich regions are often found in introns or have

a regulatory role in determining polyadenylation. Plants use G and C in the third

position of degenerate codons, A or T being rare. Bt genes have the opposite codon

usage. Codon usage is reported to be related to the abundance of the corresponding

tRNAs (Bennetzen and Hall 1982). Frequent use of rare codons would decrease the rate

of synthesis of a Cry protein in plant cells. Keeping this in view, reconstruction ofcryIIIA gene has been achieved and has shown to significantly improve the expression

(Sutton et al1992; Adang et al1993; Perlaket al1993). Additional evidences also exist

in favour of expression of other reconstructed cry genes in plants (Perlak et al 1991;

Fujimoto et al1993; Koziel et al1993; Nayaket al1995; Wunn et al1996). Bacterial cry

gene has been reported to show very high expression when made to express in

chloroplasts (McBride et al 1995). Considering the above, an argument thus can come

up on the merit of our attempts to transfer the native form of the cry gene to plants.

Native cryIA gene, when transferred to tomato was reported to confer tolerance

against highly sensitive lepidopteran insect larvae (Fischhoffet al1987; Delannay et al

1989). Additionally, a truncated form of the native cryIIIA gene with a deletion at the 5'end when driven by CaMV35S promoter could effect high expression in tomato plants

(Rhim et al 1995) and confer resistance against Colorado potato beetle. Bacterial cry

gene when used with 5'UTR regulatory elements was shown to effect high expression in

plant (Adang et al1988; Carrozi et al1992; Karet al1996). Thus, what would be the

required level of expression of the native cryIIIA gene in rice to offer the desired

protection against the target coleopteran insect pests remains to be ascertained in

further through development of transgenics.

Acknowledgements

The authors thank Drs Debabrata Basu and Sampa Das for their suggestive interac-

tions. Grants from the Rockefeller Foundation, New York (RF 89026#39) and the


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Department of Biotechnology, New Delhi to SKS; the University Grants Commission,

New Delhi to PN in the form of an Associateship and the Department of Atomic

Energy, New Delhi to ASR in the form of a Fellowship are acknowledged gratefully.

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Corresponding editor: R JAYARAMAN

clontng of a cryiiia endotoxin gen bt

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