Production Of Insecticidal Proteins Research Articles

Plant Lectins as Insecticidal Agents Against Cotton Leafworm Spodoptera littoralisand Their Potential Applications in Crop Protection

Background: The production of insecticidal proteins is one of the greatest vital direct defense responses in plants against the attack by phytophagous insects. One particular class of the entomotoxic proteins present in many plant species is lectins that is used against a wide range of important insects. Aim: The insecticidal activity of two lectins from seeds of white kidney bean (Phaseolus vulgaris) and soybean (Glycine max) on the growth, development and survival of the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied. Methods: Lectin products were isolated and purified using ammonium sulfate precipitation. Insecticidal activity, growth-inhibitory and physiological effects on the differential haemocyte count of lectin products were evaluated against S. littoralis. In vivo effects of lectin products on some S. littoralis enzymes were also determined. Results: Lectin contents in 250 g seeds were 700 and 500 mg in soybean and white kidney bean, respectively. The total protein concentrations were 160-163 and 118-120 mg/mL in soybean and white kidney bean, respectively. The insecticidal activity of lectin products exerted a strong effect on the larval weight gain and the reduction of the weight reached to 75.25%. Furthermore, the other developmental stages of pupation and adult formation were also affected. An elongated larval duration was noticed since larval duration was 24.8 days compared with control (12.0 days). Complete pupation (100%) was recorded in the control; however, a significant decrease in pupation was found to be 54.0% in treated larvae. Lectin products increased the average number of 123.9 haemocytes (123.9) compared to the control (94.4 haemocytes/field). In addition, lectin products inhibited the activity of total proteases, α-amylase, GST and total esterases. Conclusion: It can be concluded that the lectins have high potential in pest control strategies based on their activity towards the cotton leafworm, S. littoralis. These findings suggest that lectin products are interesting tools for integrated pest management in important agriculture crops.

Transgene behavior in genetically modified teosinte hybrid plants: transcriptome expression, insecticidal protein production and bioactivity against a target insect pest

BackgroundIn 2009, Spanish farmers reported a novel weed, now identified as a relative of maize’s ancestor, teosinte, in their maize fields. Introgression of the insect resistance transgene cry1Ab from genetically modified (GM) maize into populations of this weedy Spanish teosinte could endow it with additional defense mechanisms. The aims of this study were: (1) to test if hybridization between GM maize and weedy plants from Spain is possible; (2) to understand the relationship between transgene transcription activity, concentrations of the expected transgene product (Cry1Ab protein) and the bioactivity of the latter on target insect pests following transgene flow from GM maize into Spanish teosinte plants.ResultsWe demonstrated that hybridization between GM maize and the weedy Spanish teosinte is possible, with no observable barrier to the formation of crop/weed hybrids when teosinte served as pollen donor. When GM maize plants were used as pollen donors, significant crossing incompatibility was observed: hybrid plants produced only few “normal” seeds. Nevertheless, viable F1 seeds from GM pollen crossed onto teosinte were indeed obtained. The cry1Ab transgene was stably expressed as mRNA in all crossings and backgrounds. Similarly, toxicity on neonate Ostrinia nubilalis, presumably due to Cry1Ab protein, was consistently expressed in teosinte hybrids, with mortality rates 95% or higher after only 4 days exposure, similar to rates on parental GM maize plants. Nevertheless, no strong correlations were observed between transgene transcription levels and Cry1Ab concentrations, nor between Cry1Ab concentrations and insect mortality rates across all of the different genetic backgrounds.ConclusionsOur results establish fundamental parameters for environmental risk assessments in the European context: first, we show that crop/weed hybridization in fields where maize and teosinte exist sympatrically can lead to potentially catastrophic transfer of resistance traits into an already noxious weed; second, our results question the viability of using gene dosage to model and predict ecological performance in either the intended crop plant or the undesired teosinte weed. Significant questions remain that should be addressed in order to provide a scientific, sound approach to the management of this novel weed.

Combined stress of low temperature and flooding affects physiological activities and insecticidal protein content in transgenic Bt cotton

Low temperature and flooding are severe stresses to cotton plant growth and development in some parts of the world. This study was conducted to determine the interactive effects of low temperature and flooding on physiological and growth parameters of cotton plants, and yield of seed cotton. Transgenic Bt cotton plants at the boll-setting stage were treated with low temperature, flooding, and their combinations. The stresses decreased photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration by up to 37%, 71%, 52%, and 60%, respectively. The content of soluble protein and insecticidal protein was also decreased by up to 65% and 53%, respectively. By contrast, malondialdehyde (MDA) content increased by up to 114%. The length of fruiting branch was shortened by combined low temperature and flooding stress. Boll number and weight were decreased by 3.2% and 28.1%, respectively. Seed cotton yield was decreased by up to 43.2%. Our results suggest that concurrent low temperature and flooding at the boll-setting stage negatively influenced leaf photosynthetic activities and insecticidal protein production, leading to poor protection and yield. We recommend that agronomic measures be designed to stabilise physiological activities and insecticidal protein production in Bt cotton plants under stress conditions.

YmoA negatively controls the expression of insecticidal genes in Yersinia enterocolitica

Yersinia enterocolitica is toxic towards invertebrates due to the presence of the toxin complex (tc) genes that are activated by the thermolabile regulator TcaR2. In the search for further regulatory factors involved in insecticidal gene expression, the modulator of yersinial virulence, YmoA, was identified to silence all tc genes of the Y. enterocolitica strain W22703 (biovar 2, serovar O:9). Using promoter fusions with the luciferase reporter, we found that the deletion of ymoA results in elevated transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at both 15 °C and 37 °C. Complementation by episomal ymoA significantly reduced tc gene expression, thus validating the inhibitory activity of YmoA on the production of insecticidal proteins. YmoA contributes to the binding properties of H-NS to the tc promoters by forming a complex with this nucleoid-associated protein, and this complex not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB that play an important role in controlling the expression of both genes. At low temperature, the intracellular amount of thermostable YmoA is not reduced, but the repressor is less functional. These data point to H-NS/YmoA as an antagonist of the inducer TcaR2.

Efficient transcription of the larvicidal <i>cry</i>4<i>Ba</i> gene from <i>Bacillus thuringiensis</i> in transgenic chloroplasts of the green algal <i>Chlamydomonas reinhardtii</i>

Unicellular micro-alga Chlamydomonas reinhardtii has been recognized as a promising host for expressing recombinant proteins albeit its limited utility due to low levels of heterologous protein expression. Here, transcription of the 3.4-kb mosquito-larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic C. reinhardtii chloroplasts under control of the promoter and 5’-untranslated region of photosynthetic psbA gene was accomplished. Inverted repeats in chloroplast genomes of the host strain with deleted endogenous psbA genes were selected as recombination targets. Two transformant lines were obtained by dual-phenotypic screening via exhibition of resistance to spectinomycin and restoration of photosynthetic activity. Stable and site-specific integration of intact cry4Ba and psbA genes into chloroplast genomes found in both transgenic lines implied homoplasmy of organelle populations. Achievement in cotranscription of cry4Ba and psbA transgenes revealed by RT-PCR and Northern blot analyses demonstrates the sufficiency of this system’s transcription machinery, offering the further innovation for insecticidal protein production.

Production of insecticidal protein of Bacillus thuringiensis by cultivation of recombinant Escherichia coli

Abstract Cultures of recombinant Escherichia coli JM103 harboring the 135 k dalton (Da) insecticidal protein (IP) gene from Bacillus thuringiensis subsp. aizawai strain IPL7 were investigated to obtain high cell density and a large amount of 135kDa IP. In test tube cultures of recombinant E. coli JM103, soybean hydrolysate was apparently effective in improving 135kDa IP production. However, when fed-batch culture was carried out with soybean hydrolysate, a large amount of acetate accumulated in the broth and acetate was evidently the main inhibitor of cell growth and 135kDa IP production. In recombinant E. coli JM101, JM105 and JM109 producing 135kDa IP, the addition of soybean hydrolysate also increased 135kDa IP production, but it stimulated acetate accumulation as well. In order to remove acetate from the broth, cultivation with cross-flow filtration was employed. The final cell concentration of recombinant E. coli JM103 was 125 g/dm 3 , and 135kDa IP was produced at a concentration of 6.6 g/md 3 .