cry1A Gene Research Articles

Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.).

Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.

Diversity of Bacillus thuringiensis Isolates Native to Uttarakhand Himalayas, India and Their Bioefficacy Against Selected Insect Pests

The huge family status and diversified agricultural applications of Bacillus thuringiesnsis (Bt) Berliner made the bacterium, a highly specialized bio-insecticide with worldwide commercial usage. Novel strains and cry genes always expand potential usage of the bacterium. In the present investigation, the authors isolated 45 candidate strains of Bt from a unique ecological niche of Indian subcontinent, i.e., Uttarakhand state belongs to North Western Himalayas of India. All the isolates were characterized for light microscopy, PCR, SDS-PAGE and bioassays against major insect pests. The diversity in collection is manifested by existence of two major crystal shapes (Bipyramidal and spherical) and 21 different cry gene profiles with predominance of cry1 (cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1I) family genes followed by cry2 (cry2A, cry2B) and cry26. Interestingly, coleopteran specific genes were governed by cry8 not the cry3. The sequencing of major cry genes showed preferential divergence of cry2A genes and led to partial identification of novel cry1D gene from UKBt13. Majority of the isolates showed existence of 68 and 130 kDa crystal toxins. The bioefficacy studies presented here led us to identify putative novel strains with potential applications in pest management programs and associated toxicological diversity of Bt in Uttarakhand Himalayas.

Development of a Sri Lankan rice variety Bg 94-1 harbouring cry2A gene of Bacillus thuringiensis resistant to rice leaffolder [Cnaphalocrocis medinalis (Guenée)

The rice leaffolder (RLF) [ Cnaphalocrocis medinalis (Guenee)] is a major lepidopteran insect pest of rice in Sri Lanka and the larvae cause significant yield losses annually. Insecticidal crystal protein gene, cry2A driven by the 35S cauliflower mosaic virus (CaMV) promoter with a double enhancer sequence was transformed into scutellum derived embryogenic calli of Bg 94-1 via Agrobacterium tumefaciens mediated transformation procedure. Transformation vector pCAMBIA 1305.1 containing the transgene construct CaMV 35S d-cry2A-NOS, hygromycin phosphotransferase and β-glucuronidase (GUS)-Plus was employed in this study. Transformed calli were subjected to 4 ‒ 5 rounds of hygromycin selection. Molecular analysis of the generated plants together with an insect bioassay using RLF larvae confirmed successful transformation and expression of this transgene in T0 generation of rice plants. A total of 19 putative transgenic plants could be confirmed from a total of 60 T0 generation plants examined. Insect feeding bioassay demonstrated that T0 transgenic rice plants are effective against RLF larvae, and showed approximately 89 % larval mortality after 3 days and 100 % mortality after 5 days of feeding. Further molecular analysis of T1 generation of the transgenic lines confirmed the stable integration of cry2A transgene into the Bg 94-1 genome. Findings of this study demonstrated that the cry2A gene of Bacillus thuringiensis ( Bt ) was successfully transformed into Bg 94-1 via Agrobacterium mediated transformation and have developed resistance against the RLF larvae. Thus, this study highlights the importance of developing transgenic rice, which can provide insect resistance against a major lepidopteran insect pest of rice in Sri Lanka.

Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley

Bacillus thuringiensis (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, new indigenous B. thuringiensis strains were isolated and investigated so that these could be used as an alternative and/or support the current commercial strains/cry proteins in use. For this, 159 samples including soil, leaf and spider webs were collected from ten districts of Kashmir valley (India). Of 1447 bacterial strains screened, 68 Bt strains were identified with 4 types of crystalline inclusions. Crystal morphology ranking among the Bt strains was spherical (69.11%) > spore attached (8.82%) > rod (5.88%) = bipyramidal (5.88%) > spherical plus rod (4.41%) > spherical plus bipyramidal (2.94%) = cuboidal (2.94%). SDS-PAGE investigation of the spore–crystal mixture demonstrated Bt strains contained proteins of various molecular weights ranging from 150 to 28 kDa. Insecticidal activity of the 68 indigenous Bt strains against Spodoptera litura neonates showed that Bt strain SWK1 strain had the highest mortality. Lepidopteron active genes (cry1, cry2Ab, cry2Ab) were present in six Bt strains. Further, analysis of a full-length cry2A gene (~1.9 kb) by PCR–RFLP in strain SWK1 revealed that it was a new cry2A gene in Bt strain SWK1 and was named as cry2Al1 (GenBank Accession No. KJ149819.1) using the Bt toxin nomenclature (http://www.btnomenclature.info). Insect bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 is toxic to S. litura with LC50 2.448 µg/ml and H. armigera with LC50 3.374 µg/ml, respectively. However, it did not produce any mortality in third instar larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi larvae/pupae insects (100 µg/ml) at 28 ± 2 °C and 75 to 85% relative humidity under a photoperiod of 14L:10D.

Cloning and expression of cry2Aa from native Bacillus thuringiensis strain SY49-1 and its insecticidal activity against Culex pipiens (Diptera: Culicidae).

Bacillus thuringiensis (Berliner) (Bt) is well known for having toxicity against pest insects because of their ability to form endospores and broad-range activity of their parasporal inclusions. In this study, a new member of cry2A gene from previously characterized native B.thuringiensis SY49-1 strain was cloned, expressed and used for its activity against Culex pipiens (Diptera: Culicidae) larvae. The sequence analysis of the cloned cry2A gene revealed that it encodes a polypeptide of 633 aa residues with 99% identity to Cry2Aa protein with expected molecular weight of 70.7kDa. Bacillus thuringiensis delta-endotoxin nomenclature committee designed our sequence as Cry2Aa18 being a new member of Bt toxins. Bioassays against last instar larvae of C.pipiens indicated that Cry2Aa18 has considerable toxicity with LC50 of 630μgml-1. In order to prevent the spread of infectious diseases mediated by C. pipiens, this newly characterized cry2Aa18 gene could constitute as an important biological control tool for controlling mosquito larvae living in freshwater systems and can be used as a good alternative for minimizing the use of chemicals.

Effects of 24-epibrassinolide and green light on plastid gene transcription and cytokinin content of barley leaves

In order to evaluate whether brassinosteroids (BS) and green light regulate the transcription of plastid genes in a cross-talk with cytokinins (CKs), transcription rates of 12 plastid genes (ndhF, rrn23, rpoB, psaA, psaB, rrn16, psbA, psbD, psbK, rbcL, atpB, and trnE/trnY) as well as the accumulation of transcripts of some photoreceptors (PHYA, CRY2, CRY1A, and CRY1B) and signaling (SERK and CAS) genes were followed in detached etiolated barley leaves exposed to darkness, green or white light ±1μm 24-epibrassinolide (EBL). EBL in the dark was shown to up-regulate the transcription of 12 plastid genes, while green light activated 10 genes and the EBL combined with the green light affected the transcription of only two genes (psaB and rpoB). Green light inhibited the expression of photoreceptor genes, except for CRY1A. Under the green light, EBL practically did not affect the expression of CRY1A, CAS and SERK genes, but it reduced the influence of white light on the accumulation of CAS, CRY1A, CRY1B, and SERK gene transcripts. The total content of BS in the dark and under white light remained largely unchanged, while under green light the total content of BRs (brassinolide, castasterone, and 6-deoxocastasterone) and HBRs (28-homobrassinolide, 28-homocastasterone, and 6-deoxo-28-homocastasterone) increased. The EBL-dependent up-regulation of plastome transcription in the dark was accompanied by a significant decrease in CK deactivation by O-glucosylation. However, no significant effect on the content of active CKs was detected. EBL combined with green light moderately increased the contents of trans-zeatin and isopentenyladenine, but had a negative effect on cis-zeatin. The most significant promotive effect of EBL on active CK bases was observed in white light. The data obtained suggest the involvement of CKs in the BS- and light-dependent transcription regulation of plastid genes.

Effects of the sequence and orientation of an expression cassette in tobacco transformed by dual Bt genes

This study investigated the effects of the sequence arrangement and orientation of a target gene expression cassette in vectors on expression levels to determine the optimal combination for highly efficient multi-gene expression. Five plant transformation vectors were constructed using dual Bt genes, Cry1Ac and Cry3A, which differed in the sequence arrangement and orientation of the target gene expression cassette. Through an Agrobacterium-mediated method, 5 vectors were used for the genetic transformation of tobacco to obtain transgenic lines. Fluorescence quantitative PCR showed that the target genes were expressed at the transcriptional level, which did not differ significantly among the different vectors. However, an enzyme-linked immunosorbent assay showed that there were significant differences in the toxin expression levels of the different vectors. In vectors N12 and N19, the Cry1Ac gene, located upstream, showed lower average expression than the Cry3A gene, located downstream. Similarly, in vectors N13 and N18, the Cry3A gene, located upstream, had lower expression than the downstream Cry1Ac gene. For vector N21, with the expression cassette containing the Cry1Ac gene located upstream in a trans-arrangement and that of the Cry3A gene located downstream in a cis-arrangement, the Cry1Ac and Cry3A toxin levels were the highest, at 7.41 and 13.24μg·g−1, respectively. The insect resistance of transgenic lines transformed by the different vectors was related to the Bt toxin level. Resistance to H. armigera, Lepidoptera, and Cry1Ac toxin level were positively correlated; resistance to A. germari larvae, Coleoptera, and Cry3A toxin content were also positively correlated. This study showed that the sequence arrangement of 2 expression cassettes with target genes may be the key to the target gene expression. Two expression cassettes in the same orientation had little influence on gene expression; however, when the 2 expression cassettes were in the reverse arrangement, the expression of both of the target genes was promoted to a certain extent.

Development of a screening method for the monitoring of 38 genetically modified maize events in food and feed in South Korea

A multiplex PCR system was developed to trace GM maize events in food and feed. Genetic elements of 38 single GM maize events were investigated, and 10 screening targets were selected as follows: promoters [Cauliflower mosaic virus (CaMV) 35S, rice actin 1 gene (rAct1)], terminators [CaMV 35S, Agrobacterium nopaline synthase (nos), proteinase inhibitor II of potato (PinII), heat shock protein 17 of wheat (hsp17)] and structural genes [cry1A gene derived from Bacillus thuringiensis, phosphinothricin acetyltransferase gene derived from Streptomyces hygroscopicus (bar), and Streptomyces Viridochromogenes (pat), 5-enolpyruvulshikimate-3-phosphate synthase enzyme derived from Agrobacterium tumefaciens strain CP4 (cp4-epsps)]. These screening targets were combined in three individual multiplex PCR assays. The method specificity was confirmed by detecting the specific amplification of DNA derived from 16 different GM maize events. The limit of detection of the multiplex PCR system was tested using the certified reference materials of GM maize events with different GM % (w/w) and approximately 0.1%. To test the applicability of the developed screening method, 22 food samples containing maize as ingredients, and 36 maize kernels were analyzed. Various types of single and/or stacked GM events were found in 11 food and 20 maize kernel samples. These results demonstrate that the screening method developed may be useful for tracking GM maize events in food and feed.

Bioactivities of cry gene positive Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae) strains on Ephestia kuehniella Zeller, 1879 and Plodia interpunctella (Hübner, 1813) (Lepidoptera: Pyralidae)

Bacillus thuringiensis is the bacterium most commonly used for biopesticide production due to parasporal crystal formation during its growth cycle. As a consequence of repeated use, B. thuringiensis biopesticides may cause the development of resistance in the pests. Therefore, it is necessary to explore new B. thuringiensis strains with a certain degree of bioactivity. In this study (2012-2013), the bioactivity of native B. thuringiensis strains from the Aegean Region of Turkey were tested against second instar larvae of Ephestia kuehniella and Plodia interpunctella . The bioactivity of 21 B. thuringiensis strains with cry1 , cry2 or cry9 gene was determined as percent mortality according to Abbott’s formula . The highest mortality rates were 42 and 63% in E. kuehniella and P. interpunctella , respectively. These mortality rates were equal to or 1.8 times greater than that of B. thuringiensis subsp. kurstaki . In addition, plasmid profiles of B. thuringiensis strains changed between 5-18 kb . Moreover, SDS-PAGE analysis of the most toxic strains indicated the presence of Cry1 and Cry2 proteins . Two different cry2 gene profiles containing either cry2Aa1 or combination of cry2Aa1 and cry2Ab2 genes were detected by PCR analysis . In addition, partial DNA sequence analysis of cry2A genes indicated phylogenetic differences among the toxic strains and B. thuringiensis subsp. kurstaki . As a result, these B. thuringiensis strains may be used to control both E. kuehniella and P. interpunctella as alternative biopesticides in cases of insect resistance to currently used B. thuringiensis preparations.

Genetic transformation and expression of Cry1Ac–Cry3A–NTHK1 genes in Populus × euramericana “Neva”

To realize the genetic transformation of Cry1Ac, Cry3A, and NTHK1 genes in Populus × euramericana “Neva” and obtain transgenic plants, the plant transformation vector p09687199–Cry1Ac–Cry3A–NTHK1 was transformed in Populus × euramericana “Neva” through Agrobacterium-mediated transformation. The complete regeneration of the transformed plant was screened with kanamycin. Resistant roots of the transformed plants were detected through PCR, fluorescence quantitative PCR, and ELISA analysis of the toxic protein. The insect resistance and salt tolerance of transgenic plants were determined based on the findings of these methods. PCR detection showed that among six lines, five lines indicated the presence of three target genes, Cry1Ac and Cry3A genes were detected in one line. Fluorescence quantitative PCR detection showed that the transcript abundance of the Cry1Ac gene was within 1.13E+3 to 3.17E+4, that of Cry3A was within 4.40E+6 to 1.97E+7, and that of NTHK1 was within 2.48E+3 to 8.17E+3. Bt toxic protein detection showed that the content of the Cry1Ac gene was from 1.08 to 22.99 ng·g−1, and that of Cry3A gene was from 146.12 to 17027.96 ng·g−1. The expression of the Cry3A gene was significantly higher than that of the Cry1Ac gene. The insect resistance test demonstrated that the mortality of transgenic plants on Hyphantria cunea first instar larva reached 68.89 %. The mortality on Plagiodera versicolora first and second instar larvae reached 100 %, whereas that on the third instar larva was as high as 81.71 %. Four lines were selected to detect potting salt tolerance, and only the No. 2 line showed strong salt tolerance.

Screening of new isolates of Bacillus thuringiensis for cry1 genes and testing of toxicity against Dichocrocis punctiferalis (Family: Pyralidae, Order: Lepidoptera)

In order to identify the more toxic novel cry gene, the cry1 gene was screened in six indigenous isolates of Bt by PCR with degenerate primers showed amplification in all the Bt isolates. Subsequent screening of cry1 subfamily gene(s) by gene specific primer showed amplification of cry1A gene in the five Bt isolates, three out of the six cry1 positive isolates showed the presence of cry1Aa gene. One of the six Bt isolates showed the presence of cry1Ab gene. Five Bt isolates showed amplification for cry1Ac gene and a variation in size of amplification was observed in one of the Bt isolates Bt, T27. Further, SDS-PAGE analysis of a spore crystal mixture isolated from new isolates of Bt, T27 showed a single band of ~135 kDa indicating presence of cry1Ac gene. The toxicity analysis of Bt strain T27 against Dichocrocis punctiferalis showed 100 per cent mortality on the fifth day after treatment. The varied ~925 bp amplicon of cry1Ac gene of Bt, T27 was amplified and cloned in a T/A vector. Comparison of nucleotide sequence data generated from the cry1Ac (~925 bp) gene showed 99 percent homology and two amino acid variation when comparison with its holotype sequence of Cry1Ac1.

Cloning, characterization and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1, native to Himalayan valley Kashmir

Bacillus thuringiensis (Bt) is a gram positive bacterium which is effectively being used in pest management strategies as an eco-friendly bioinsecticide. In the present study a new cry2A gene was cloned from a promising indigenous B. thuringiensis SWK1 strain previously characterized for its toxicity against Spodoptera litura and Helicoverpa armigera larvae. The nucleotide sequence of the cloned cry2A gene pointed out that the open reading frame has 1902 bases encoding a polypeptide of 634 amino acid residues with a probable molecular weight of 70kDa. Homology comparisons showed that the deduced amino acid sequence of Cry2A had a similarity of 94% compared to that of the known Cry2Aa protein in the NCBI database and this gene has been named as cry2Al1 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry2Al1 was ligated into pET 22b vector and expressed in Escherichia coli BL21 (DE3) pLysS under the control of T7 promoter induced by isopropyl-beta-d-thiogalactopyranoside (IPTG). SDS-PAGE analysis confirmed the expression of cry2Al1 as ∼65kDa protein. Insect pest bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 are toxic to S. litura and H. armigera with LC50 2.448μg/ml and H. armigera with 3.374μg/ml respectively.

Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac + Cry2A, GT gene and insect mortality

Expression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production.

Effect of dual Bt-expression transformation vectors on transgene expression in tobacco.

This study aimed to determine the influence of vector structure on dual Bt gene expression and establish an efficient expression vector using Cry1Ac and Cry3A genes. Four vectors (N4, N5, N10, and S23) were developed and used for genetic transformation of tobacco to obtain insect-resistant transgenic lines. The vectors were constructed using the MAR structure, applying different promoter and enhancer sequences, and changing the transgene open-reading frame sequence. The average Cry1Ac toxalbumin expression quantity was 67 times higher in N5 than in N4 transgenic lines (8.77 and 0.13 μg/g, respectively). In contrast, the average Cry3A toxalbumin expression quantity was 1.5 times higher in N4 than in N5 lines (12.70 and 8.21 μg/g, respectively). The sequences of both Bt genes significantly influenced toxalbumin expression, although upstream Bt genes presented lower expression levels. The average Cry1Ac toxalbumin content was 13 times higher in the transgenic lines of AtADH 5'-non-translated sequence N5 (8.77 mg/g) than in the omega N10 lines (0.67 mg/g). Furthermore, the average Cry1Ac toxalbumin content was 5 times higher in MAR N5 than in non-MAR S23 lines (8.77 and 1.63 mg/g, respectively). The average Cry3A toxalbumin content was 1.3 times higher in N5 than in S23 lines (8.21 and 6.48 mg/g, respectively). Moreover, toxalbumin expression levels differed significantly among the S23-transformed lines. The MAR structure applied on both ends of the genes increased both the level and stability of exogenous gene expression. In conclusion, N5 was the most optimal of the four tested vectors.

Molecular Cloning of Two Maize ( Zea mays ) CRY1a Genes and Their Expres-sion Patterns of in Response to Different Light Treatments

Molecular Cloning of Two Maize ( <i>Zea mays</i> ) CRY1a Genes and Their Expres-sion Patterns of in Response to Different Light Treatments

Genetic transformation and expression of transgenic lines of Populus x euramericana with insect-resistance and salt-tolerance genes.

We characterized new transgenic varieties of poplar with multiple insect-resistant and salt stress tolerant genes. Two insect-resistant Bacillus thuringiensis (Bt) genes, Cry1Ac and Cry3A, and a salt-tolerant gene, Betaine aldehyde dehydrogenase (BADH) were inserted into a vector, p209-Cry1Ac-Cry3A-BADH. The clone of Populus x euramericana was transformed by the vector using the Agrobacterium-mediated method. Three transgenic lines were assessed using genetic detection and resistance expression analysis. PCR revealed that exogenous genes Cry1Ac, Cry3A, BADH and selective marker gene NPTII were present in three transgenic lines. Quantitative real-time PCR (qPCR) showed significant differences in the transcriptional abundance of three exogenous genes in different lines. Results of assays for Bt toxic proteins showed that the Cry1Ac and Cry3A toxic protein content of each line was 12.83-26.32 and 2108.91-2724.79 ng/g, respectively. The Cry1Ac toxic protein content of different lines was significantly different; the Cry3A toxic protein content was about 100 times higher than that of the Cry1Ac toxic protein. The insect-resistance test revealed the mortality rate of transgenic lines to Hyphantria cunea L1 larvae varied by 42.2-66.7%, which was significantly higher than non-transgenic lines. The mortality rate of L1 and L2 Plagiodera versicolora larvae was 100%. The insecticidal effect of transgenic lines to P. versicolora larvae was higher than that to H. cunea larvae. NaCl stress tolerance of three transgenic lines under 3-6% NaCl concentration was significantly higher than that of non-transgenic lines.

A rapid loop-mediated isothermal amplification method for detection of the modified GM cry1A gene in transgenic insect-resistant cotton and rice

Among the commercial genetically modified (GM) crops, the insect-resistant GM crops are the major cultivars that cry gene is introduced into. A cry1Ab/1Ac GM fusion gene (GFM cry1A) and a GM truncated cry1Ac gene (cry1Ac-Mon) is the key foreign gene employed for construction of GM crops by China researchers and Monsanto Technology LLC respectively. Here these two genes are entitled “GM cry1A” gene and a rapid visual loop-mediated isothermal amplification (LAMP) assay method for detection of GM cry1A in transgenic insect-resistant crops was established. The LAMP assay was performed at an optimal temperature of 65 °C for 60 min in the presence of a set of four specific primers recognized six distinct sequences of the GM cry1A gene. The rough detection limit to the GM cry1A in samples is as low as 0.01% (a weight ratio of transgenic insect-resistant rice/cotton to non-transgenic rice/cotton). Comparatively, the sensitivity of this LAMP method is 10 times over that of the conventional PCR method. Fifteen cultivars/events and five Bt strains with or without cry1A gene were analyzed using the LAMP method as well as PCR method. The results demonstrate that this LAMP method shows a distinct specificity to the GM cry1A gene comparing with PCR analysis. Therefore, this LAMP method will be a potential effective tool for screening the GM cry1A gene in GM crops which are widely plant in China and other developing countries.

Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis

Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis

Cry Proteins from Bacillus thuringiensis Active against Diamondback Moth and Fall Armyworm.

Biopesticides based on Bacillus thuringiensis and genetically modified plants with genes from this bacterium have been used to control Plutella xylostella (L.) and Spodoptera frugiperda (J.E. Smith). However, the selection pressure imposed by these technologies may undermine the efficiency of this important alternative to synthetic insecticides. Toxins with different modes of action allow a satisfactory control of these insects. The purpose of this study was to characterize the protein and gene contents of 20 B. thuringiensis isolates from soil and insect samples collected in several areas of Northeast Brazil which are active against P. xylostella and S. frugiperda. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Polymerase chain reaction assays were used to determine toxin genes present within bacterial isolates. The protein profile of the majority of the isolates produced bands of approximately 130kDa, suggesting the presence of Cry1, Cry8 and Cry9 proteins. The gene content of the isolates of B. thuringiensis investigated showed different gene profiles. Isolates LIIT-4306 and LIIT-4311 were the most actives against both species, with LC50 of 0.03 and 0.02 × 10(8)sporesmL(-1), respectively, for P. xylostella, and LC50 of 0.001 × 10(8)sporesmL(-1) for S. frugiperda. These isolates carried the cry1, cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry2, cry2A, cry8, and cry9C genes. The obtained gene profiles showed great potential for the control of P. xylostella and S. frugiperda, primarily because of the presence of several cry1A genes, which are found in isolates of B. thuringiensis active against these insects.

Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle

The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47mg/larvae/day and 12.46mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China.