Pyramiding of cry toxins and methanol producing genes to increase insect resistance in cotton

Abstract

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve insect pest control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus. niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety by using shoot apex-cut Agrobacterium-mediated transformation. Expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, in contrast to the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes showed 100% mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for Pectinophora gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes are an effective strategy to improve the control of different insect pests.

Keywords: Cotton; agrobacterium; eagle-2; insecticidal cry proteins; pectin methyl esterase enzyme; transformation.

Figure 1.

Restriction analysis of puc57 vectors containing the cry1Fa and cry32Aa gene cassette (total 7954 bp size) or the AtPME and AnPME gene cassette (total 7893 bp size). Figure 1a, BamHI and EcoRI restriction analysis of the cry1Fa and cry32Aa gene cassette. M: 10kb ladder. Lane 2: cry1Fa and cry32Aa gene cassette positive sample. Lane 1: negative control of pUC57 without insert samples. Figure 1b, EcoRI and HindIII restriction analysis of the AtPME and AnPME gene cassette. M: 10 Kb ladder. Lane 1, 2: AtPME and AnPME gene cassette positive samples. Figure 1c, schematic representation of both gene cassettes containing cry1Fa and cry32Aa genes or AtPME and AnPME genes, respectively

Figure 2.

Restriction digestion and PCR analysis of both cry genes and pme genes cassettes

Figure 3.

PCR amplification of both cry genes and pme genes from transformed A. tumefaciens with the corresponding pCAMBIA1301-cryCassette and pCAMBIA1301-pmeCassette constructions

Figure 4.

Determination of cry genes and PME genes expression in transgenic cotton plant by PCR analysis

Figure 5.

Relative expression of cry and PME genes in four transgenic cotton plants (K1, K2, K3 and K4)

Figure 6.

Methanol quantification in transgenic cotton plants. Standard curve of methanol was done by Mass Spectrometry (MS) with reference to a methanol standard. The transgenic cotton plants, namely, K1, K2, K3, K4 were subjected to methanol quantification

Figure 7.

Cotton leaf chewing and mortality assay of H. armigera in different transgenic cotton plants

Figure 8.

Pink Bollworm P. gossypiella mortality assays in different transgenic cotton plants. Figure 8a and b, in non-transgenic cotton plants, the cotton boll showed to be completely damaged by P. gossypiella and and larvae that were still alive are highlighted with a red circle mark. Figure 8c and d, in transgenic plants transformed with the double Bt-gene (cry1Fa and cry32Aa), the plant bolls showed insect resistance and only one locule was damaged but P. gossypiella larvae were dead. Figure 8e and f in the transgenic plants transformed with double Bt-gene (cry1Fa and cry32Aa) and double AnPME and AtPME gene in the same plant; the plants showed full resistance and even spotted was found dead on leaf and P. gossypiella free cotton was fully developed

Figure 9.

Mortality analysis against army boll worm Helicoverpa armigera, Pink boll worm Pectinophora gossypiella and spotted bollworm Earias fabia. Two-way ANOVA showed the significance of the data. compared to the non-transformed plants