Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Diversidad genético molecular de cepas de Bacillus thuringiensis con potencial tóxico contra Aedes aegypti / Genetic and molecular diversity of Bacillus thuringiensis strains and insecticidal activity potential against Aedes aegypti

Objetivos: Evaluar la diversidad genético molecular de cepas nativas aisladas de B. thuringiensis tóxicas contra Aedes aegypti, vector del dengue. Diseño: Estudio descriptivo, analítico. Institución: Centro de Investigación de Bioquímica y Nutrición, Facultad de Medicina, UNMSM. Material biológico: Cepas nativas y estándar de B. thuringiensis. Intervenciones: Se extrajo el ADN genómico de 53 cepas nativas y 10 cepas estándar de B. thuringiensis aisladas del suelo de distintas regiones geográficas del Perú. Para el análisis de diversidad, se evaluó secuencias repetitivas de ADN mediante la técnica REP-PCR, siendo visualizados en geles de agarosa teñidos con bromuro de etidio. Se hizo el análisis de clúster con el dendograma de similaridad de cepas estándares y nativas de B. thuringiensis, utilizando programas bioinformáticos. Principales medidas de resultados: Diversidad evaluada mediante los perfiles genéticos (bandas de ADN repetitivas) en geles de agarosa y dendograma de cepas B. thuringiensis. Resultados: Las cepas procedentes de Junín, Huaral, Ica, Cusco, Arequipa y Cajamarca tendieron a formar grupos según procedencia, destacándose dos caracterizadas como potencialmente tóxicas contra Aedes aegypti (de Ica y de Cajamarca); estas muestran el gen cry2 y cry4 (datos no presentados), formando un subgrupo con cepas estándar tóxicas HD-968 y GM 33, las que contienen el gen cry2. Conclusiones: Se observa gran diversidad de cepas nativas de B. thuringiensis procedentes de diferentes lugares del país, con cierta tendencia a formar subgrupos según procedencia geográfica y en relación de similaridad con las cepas B. thuringiensis estándares, algunas con potencial para ser utilizadas contra Aedes aegypti, vector del virus del dengue.

Objectives: To determine the molecular genetic diversity of native B. thuringiensis strains toxic against Aedes aegypti, vector of dengue. Design: Descriptive, analytical study. Setting: Biochemistry and Nutrition Research Center, Faculty of Medicine, San Marcos University, Lima, Peru. Participants: Native and standard strains of B. thuringiensis. Interventions: Extraction of B. thuringiensis genomic DNA from 53 native strains and 10 standard strains isolated from different Peruvian regions soil samples. Diversity was determined by repetitive DNA sequences using REP-PCR technique, visualized in agarose gels stained with ethidium bromide. B. thuringiensis standard and native strains cluster analysis was performed by dendrogram of similarity using bioinformatic programs. Main outcome measures: Diversity of B. thuringiensis strains. Results: Strains from Junin, Huaral, Ica, Cusco, Arequipa, and Cajamarca tended to form groups according to source, highlighting two strains characterized as potentially toxic against Aedes aegypti (Ica and Cajamarca), that presented cry4 and cry2 gene (data not shown), outlining a subgroup or cluster with toxic standard strains HD-968 and GM 33 containing the cry2 gene. Conclusions: A great diversity of B. thuringiensis native strains coming from different regions of the country have a tendency to form sub-groups according to geographical origin and in relation to standard strains, some of them with a bioinsecticidal potential against B. Thuringiensis, vector of dengue.

Two new Brazilian isolates of Bacillus thuringiensis toxic to Anticarsia gemmatalis (Lepidoptera: Noctuidae) / Dois novos isolados brasileiros de Bacillus thuringiensis tóxicos para Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Bacillus thuringiensis is a bacterium used for biopesticides production and pest-resistant plants due to the synthesis of protein crystals by cry genes, which are effective in controlling several insect orders such as Lepidoptera. This work aimed at the evaluation and characterisation of two new B. thuringiensis isolates active against A. gemmatalis (Hübner 1818) larvae, which is the soybean major pest. The results showed that Bt117-4 isolate amplified fragments corresponding to cry2 and cry9 genes, and synthesised protein fragments equivalent to 130, 90 and 45 kDa. The Bt3146-4 isolate amplified DNA fragments corresponding to cry9 gene and synthesised protein fragments of 70, 58 and 38 kDa. Transmission electron microscopy revealed the presence of protein crystals in both isolates. CL50 with Cry purified proteins from Bt117-4 and Bt3146-4, corresponded to 0.195 and 0.191 µg larvae-1, respectively. The two B. thuringiensis isolates selected in this study were effective to control velvetbean caterpillar at laboratory conditions. Field tests should be carried on to develop new biopesticides formulation as well for cry genes resource for Anticarsia gemmatalis resistant transgenic plants.

Bacillus thuringiensis é uma bactéria utilizada na produção de biopesticidas e de plantas resistentes às pragas por causa da síntese de cristais proteicos pelos genes cry, os quais são eficazes no controle de diversas ordens de insetos, como os lepidópteros. O presente trabalho objetivou a avaliação e a caracterização de dois novos isolados de B. thuringiensis ativos contra lagartas de A. gemmatalis (Hübner 1818), que é a principal praga da cultura da soja. Os resultados obtidos revelaram que o isolado Bt117-4 amplificou fragmentos correspondentes aos genes cry2 e cry9, sendo que os fragmentos proteicos sintetizados foram equivalentes a 130, 90 e 45 kDa. O isolado Bt3146-4 amplificou fragmentos de DNA que correspondem ao gene cry9 e sintetizou fragmentos proteicos de 70, 58, e 38 kDa. Os dados de microscopia eletrônica de transmissão revelam a presença de cristais proteicos em ambos os isolados. A CL50, com proteínas Cry purificadas de Bt117-4 e Bt3146-4, correspondeu a 0,195 e 0,191 µg lagarta-1, respectivamente. Os dois isolados de B. thuringiensis selecionados neste trabalho mostraram-se eficientes no controle da lagarta-da-soja em laboratório, sendo recomendada sua avaliação a campo para posterior aplicação na formulação de biopesticidas ou como fonte de genes cry para a obtenção de plantas geneticamente modificadas resistentes à Anticarsia gemmatalis.

Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.