Cry1Ac Protein Expression Research Articles

Cry toxin expression in different plant parts of Bt cotton at different phenological stages

BackgroundCompared to Bollgard-I, the utilization of Cry2Ab protein in Bollgard-II cotton cultivars enhances insect control. Field and laboratory studies demonstrated reduction in the numbers of bollworm-infested terminals, squares, and bolls in Bollgard-II cotton lines when compared to both Bollgard-I and non-Bollgard cotton cultivars. This indicates that the combination of Cry2Ab with Cry1Ac increased the overall expression of proteins. The expression of Cry protein varied across different plant parts, such as leaves, bracts, squares, and bolls. As the season progresses, the expression of Cry protein decreased in these plant parts. Leaves exhibited the highest levels of Cry protein expression, followed by squares, flowers, and bolls. Variation in the expression levels of delta endotoxins in different plant parts was one of the contributing factors to the survival of pest populations on Bt cotton.ResultsUsing a commercially available QL 96 ELISA plate kit, the concentration of delta endotoxin in various plant parts at different phenological stages was determined in twelve BG-II cotton hybrids, namely Ajeet-155, JKCH-2245, RCH-3863, NCS-866, MRC-7373, JKCH-99, MRC-7387, NCEH-21, ANKR-3324, NCSI-1904, and NCHB 9902. Cry1Ac and Cry2Ab protein levels were determined from samples of flowers and fruiting parts (Rind, locule, seed, locule wall and seed) at 40, 75, 100 and 125 days after sowing (DAS) over two consecutive years 2018–2019 and 2019–2020. Cry1Ac protein content and expression was the highest at 100DAS in locule, seed and rind; followed by rind and locule wall and seed in green bolls at 125 DAS; followed by seed, locule, rind and flowers at 75 DAS; followed by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry2Ab protein content and expression was the highest at 125DAS in locule wall and seed and rind, followed by seed, locule and rind in green bolls at 100 DAS, followed by seed, locule, rind and flowers at 75 DAS, then by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry1Ac protein expression was less in comparison with Cry2Ab.ConclusionsThe research findings indicate that the locule of BG-II cotton plants exhibited the highest expression levels of Cry1Ac and Cry2Ab proteins, followed by the seeds, rind, locule wall, and flowers. These results provide valuable insights into the distribution of Cry protein expression in different plant parts, which can contribute to a better understanding of insect control in Bt cotton cultivars.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton.

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Correlation of Cry1Ac mRNA and protein abundance in transgenic Gossypium hirsutum plant.

The online version contains supplementary material available at 10.1007/s13205-021-02828-2.

Genetic improvement of Bacillus thuringiensis against the cotton bollworm, Earias vitella (Fab.) (Lepidoptera: Noctuidae), to improve the cotton yield in Pakistan

In the present study, CryIAc (Crystal protein) gene was cloned under Cry3A promoter, which is known to express vegetatively as a sporulation independent promoter, transformed into acrystalliferous strain of Bacillus thuringiensis. Its potential was evaluated as a strain to be used for formulation development against the spotted cotton bollworm Earias vitella (Fab.) (Lepidoptera: Noctuidae), the major pest of cotton, the important cash crop in Pakistan. The crystal protein produced in a new strain, i.e., Bt1, had comparable potency as of crystal protein produced during sporulation. The new strain Bt1 had the maximum expression of Cry1Ac protein during the vegetative stage, i.e., in the first 24 h, thereby reducing the growth period until crystal protein production, from 72 to 24 h. In addition, crystal protein produced during vegetative stage under Cry3A promoter remained encapsulated within the cells, which could protect them from UV degradation and was independent of sporulation; therefore; it did not produce spores and thus is not a source to spread Bt spores in the environment. It can, therefore, be called as an environmentally friendly pesticide to control cotton pest especially bollworms and ultimately improve the yield of cotton.

Efficacy of Cry1Ac protein against gypsy moth and fall webworm in transgenic poplar (Populus davidiana × Populus bolleana) by bioassay

A modified Bacillus thuringiensis (Bt) cry1Ac gene was introduced into poplar ‘Shanxin’ (Populus davidiana × Populus bolleana) by Agrobacterium-mediated transformation. Seventeen cry1Ac transgenic lines were regenerated. The integration and expression of the cry1Ac gene in these transgenic lines were analyzed by polymerase chain reaction (PCR) and reverse-transcription quantitative PCR, respectively. Variable amounts of cry1Ac mRNA accumulated in different transgenic plants. The expression of Cry1Ac toxin protein in transgenic plants was analyzed by enzyme-linked immunosorbent assay, the concentration ranged from 0.12 to 21.00 μg g−1. The concentration of Cry1Ac protein was highly consistent with mRNA amount in each transgenic line. Leaf section bioassays were conducted using the larvae of both gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). Those plants, except lines 10 and 11, caused rapid mortality of all gypsy moth and fall webworm larvae with almost no defoliation, while lines 10 and 11, with Cry1Ac protein concentrations of 0.12 and 0.76 μg g−1, respectively, provided relatively weak insect protection. Results of insect bioassays indicate that Cry1Ac protein expression over 1.3 μg g−1 consistently produced high insect mortality for both species of moths. The correlation between concentration of Cry1Ac protein and insect mortality is important to understand for managing insect pests with transgenic plants. Transgenic poplars with high insect resistance will be useful tools for managing Lepidoptera pests of these trees.

Development of transgenic tobacco lines with pyramided insect resistant genes.

Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.

A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis.

Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σE and σK. Here, we report a novel σE-dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P 5014 -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P cry8E . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P 5014 -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P cry8E -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.

The Relationship between Insect Resistance and Tree Age of Transgenic Triploid Populus tomentosa Plants.

To explore the stability of insect resistance during the development of transgenic insect-resistant trees, this study investigated how insect resistance changes as transgenic trees age. We selected 19 transgenic insect-resistant triploid Populus tomentosa lines as plant material. The presence of exogenous genes and Cry1Ac protein expression were verified using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. The toxicity for Clostera anachoreta and Lymantria dispar was evaluated by feeding fresh leaves to first instar larvae after the trees were planted in the field for 2 years and after the sixth year. Results of PCR showed that the exogenous genes had a long-term presence in the poplar genome. ELISA analyses showed significant differences existed on the 6-year-old transgenic lines. The insect-feeding experiment demonstrated significant differences in the mortality rates of C. anachoreta and L. dispar among different transgenic lines. The average corrected mortality rates of C. anachoreta and L. dispar ranged from 5.6–98.7% to 35.4–7.2% respectively. The larval mortality rates differed significantly between the lines at different ages. Up to 52.6% of 1-year-old transgenic lines and 42.1% of 2-year-old transgenic lines caused C. anachoreta larval mortality rates to exceed 80%, whereas only 26.3% of the 6-year-old transgenic lines. The mortality rates of L. dispar exhibited the same trend: 89.5% of 1-year-old transgenic lines and 84.2% of 2-year-old transgenic lines caused L. dispar larval mortality rates to exceed 80%; this number decreased to 63.2% for the 6-year-old plants. The proportion of 6-year-old trees with over 80% larval mortality rates was clearly lower than that of the younger trees. The death distribution of C. anachoreta in different developmental stages also showed the larvae that fed on the leaves of 1-year-old trees were killed mostly during L1 and L2 stages, whereas the proportion of larvae that died in L3 and L4 stages was significantly increased when fed on leaves of 6-year-old trees. Results of correlation analysis showed there was a significant correlation between the larvae mortality rates of trees at different ages, as well as between Cry1Ac protein contents and larvae mortality rates of 6-year-old trees.

High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2–2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4–5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton

Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept ‘less intrusive’ expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L−1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T 1 transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100% larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

Multivariate analysis linkage of phyllospheric microbial community of transgenic cotton from SGK321 to Cry1Ac: A temporal expression dynamics

The effects of transgenic plant on ecological and food biosafety have attracted considerable attention recently due to the exploding number of commercialization genetic modified plants. Here, we examined the changes of bacterial and fungal community compositions in the phyllosphere of Cry1Ac Bacillus thuringiensis (Bt) cotton SGK321 by molecular fingerprint method at four major growth stages. The phyllospheric bacteria diversity of SGK321 was decreased at budding stage only, indicated by Shannon index. A slight increase of diversity and abundance of fungal community could be detected at different sampling stage except the seeding stage. Fungal community showed a tendency to be affiliated with the expression of Cry1Ac protein which was detected by enzyme-linked immunosorbent assay (ELISA) method at both the seeding and budding stages. Results suggested that, compared to bacteria, fungi in phyllosphere of transgenic cotton SGK321 might be more susceptible to Cry1Ac protein, which includes Cochliobolus sp., Setosphaeria sp., Blakeslea trispora, Cryptococcus neoformans and Cryptococcus sp. Furthermore, the seasonal and temporal parameters during the growth stages were proved to be the most important factor relating with the shift of phyllospheric microorganism communities.

Agrobacterium-mediated genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] for resistance to legume pod borer Helicoverpa armigera

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L−1 kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L−1 sucrose and 0.5 mg L−1 indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6-72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.

Agrobacterium-mediated transformation of spinach (Spinacia oleracea) with Bacillus thuringiensis cry1Ac gene for resistance against two common vegetable pests

Agrobacterium-mediated genetic transformation was applied to produce transgenic plants of spinach (Spinacia oleracea) resistant to 2 pest species, Trichoplusia ni and Autographa nigrisigna. Leaf segments from in vitro spinach plants of cultivar 'Glory' were co-cultivated with A. tumefaciens strain EHA105, which harbored the plasmid pBE2111FMB containing a synthetic cry1Ac gene encoding an insecticidal crystal protein of Bacillus thuringiensis, and neomycin phosphotransferase II and bialaphos resistance genes as selectable marker genes. Kanamycin (Km)-resistant calluses were obtained from co-cultivated leaf segments 1 month after selection on 2.5 g l � 1 gellan gum-solidified MS medium containing 3 0gl � 1 sucrose, 0.5 mg l � 1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l � 1 kinetin, 100 mg l � 1 Km and 20 mg l � 1 meropenem trihydrate. Regeneration of adventitious shoots from Km-resistant calluses was performed on 2.5 g l � 1 gellan gum-solidified MS medium containing 30 g l � 1 sucrose, 0.01 mg l � 1 2,4-D, 1 mg l � 1 kinetin, 1 mg l � 1 gibberellic acid, 100 mg l � 1 Km and 10 mg l � 1 meropenem trihydrate at 14°C. PCR and Southern blot analyses of genomic DNA isolated from T1 plants confirmed the successful integration of T-DNA into the plant genome. Expression of Cry1Ac protein was confirmed in leaves of transgenic plants by Western blot analysis. Insect bioassays against T. ni and A. nigrisigna performed with T1 plants showed more than 93.3% insect mortality within 1 week. These results suggest that the cry1Ac gene was effectively expressed in spinach.

Production of mRNA from the cry1Ac transgene differs among Bollgard® lines which correlates to the level of subsequent protein

Commercial cultivars of Bollgard cotton, Gossypium hirsutum L., differ in the amount of expressed Cry1Ac protein. However, the plant-mechanism for which this occurs is still unknown. Using quantitative real-time polymerase chain reaction (qPCR), we developed a method to determine if differences in the overall level of Cry1Ac among Bollgard lines could be correlated to the mRNA transcripts. Our data shows that the cry1Ac mRNA transcript differs among Bollgard lines and are correlated with corresponding Cry1Ac protein levels. In addition, qPCR based methods can efficiently be employed to quantify Cry1Ac protein expression levels in transgenic cotton cultivars. We postulate that qPCR based methods could be successfully employed for quantifying expression levels of transgenes in plants carrying different Bt toxins.

Expression of Cry1Ac protein in cry1Ac/CpTI transgenic rice and its resistance in different developmental stages to Chilo suppressalis

AbstractThe expression patterns of Cry1Ac protein and the resistance to striped stem borer Chilo suppressalis (Walker) of cry1Ac/CpTI transgenic rice (Oryza sativa L.) were investigated by ELISA and laboratory bioassay. ELISA results showed that Cry1Ac protein could be detected in all organs during the development process of cry1Ac/CpTI transgenic rice. The content of Cry1Ac protein varied markedly in different tissues at different stages: it was higher in the earlier stage than in the later stage. The highest level of Cry1Ac protein in terminal leaves of the main stem was found at the jointing stage. Thereafter, it decreased at the booting stage, and a gradual reduction to the lowest level was recorded from the flowering to the filling stage, before a slight increase was shown at the maturing stage. In addition, higher concentrations of this protein were detected in the main stem at the seedling, jointing and flowering stages than at other stages. Relatively lower levels of Cry1Ac protein were found in seeds. The results from laboratory bioassay showed high resistance of terminal leaves of the main stem in all developmental stages to C. suppressalis larvae. Corrected mortality values of C. suppressalis at 4 days after feeding on the detached leaves of cry1Ac/CpTI transgenic rice reached 90% before the filling stage and decreased to nearly 60% after the filling stage. Interestingly, results of whole plant bioassay coincided with those obtained in the detached leaf bioassay. The tendency of larval morality in the detached leaf bioassay and whole plant bioassay was consistent with that of Cry1Ac protein content in the different growing periods and organs.

Signal peptide of potato PinII enhances the expression of Cry1Ac in transgenic tobacco.

The modified Cry1Ac was expressed in transgenic tobacco plants. To allow secretion of the Cry1Ac protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the Cry1Ac encoding region. Expression of Cry1Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry1Ac protein and led to the secretion of the Cry1Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.