Characterizaiton, Cloning and Expression of Bt Gene Against Cotton Pests

Abstract

The efficacy of Bt toxins is diminishing because insects are becoming resistant to Bt δ-endotoxins. Second generation Bt vegetative insecticidal proteins (VIPs) could be a possible alternate of Bt crystal proteins. It has been demonstrated that Vip3Aa do not share any sequence similarity with any known Cry proteins and bind with different receptors sites in insect midgut. Plant lectins are carbohydrate-binding proteins that specifically recognize glycan structures in brush border membrane vesicle receptors present on gut epithelial cells of insects. In this study, codon optimized synthetic Bt Vip3Aa gene (2370 bp) driven by constitutive CaMV35S promoter and Allium sativum leaf agglutinin (ASAL) gene (339 bp) under phloem-specific RTBV promoter in a single 4870 bp (Vip3Aa+ASAL) cassette cloned in plant expression vector pCAMBIA 1301 under XhoI and HindIII were transformed in a local cotton variety (CEMB-33). For this purpose, the recombinant pCAMBIA_Vip3Aa+ASAL plasmid was electroporated in Agrobacterium tumefaciens strain LBA 4404 and subjected to inoculation with injured cotton embryos for introduction of desired genes through Agrobacterium to develop high resistance against major chewing and sucking insects. The putative transgenic cotton plants were confirmed through PCR by using gene-specific primers. The amplification of 587 bp fragment of ASAL gene and 682 bp fragment of Vip3Aa confirmed the successful introduction of desired genes in cotton. Total eighteen plants were found positive out of total fifty-three plants that were shifted in the field. The transformation efficiency was found to be 1.17%. The transgenic plants were also screened through Vip3Aa specific dipsticks and expression of GUS marker gene. The mRNA expression of both the genes was studied in five T1 transgenic cotton lines through quantitative real-time PCR (qRT-PCR). The comparative analysis of Ct values obtained by qRT-PCR analysis revealed that the mRNA expression of Vip3Aa gene varied from 2-8.7 folds in the lines L3P2 and L6P3 respectively. Similarly, the comparison of Ct values showed that the mRNA expression of ASAL gene ranged between 2-5 folds in transgenic lines L4P4 and L34P2. The transgenic line L6P3 showed significantly higher expression for both the genes. The expression of Vip3Aa protein in T0 and T1 generations was quantified through ELISA. The maximum protein concentration in both generations was seen in L6P3. The transgene location and copy number in the cotton genome were determined through Fluorescence in situ hybridization (FISH) in T2 generation. The transgenic cotton plant from transgenic line L3P2 showed homozygosity (two copy numbers) on chromosome number 9 at late telophase stage and one copy number at chromosome number 10 at prophase stage. Different morphological and physiological parameters of transgenic cotton lines in T1 progeny were studied in comparison to non-transgenic cotton plants. Statistically significant variation was observed in transgenic cotton lines for all the studied morphological characteristics, whereas, no significant differences were observed for physiological parameters. Similarly, the scanning electron microscopic (SEM) images of transgenic and non-transgenic cotton plants showed no visible difference in fiber morphology between transgenic and non-transgenic cotton plants which depicts no positive or negative correlation between expression of insecticidal genes and cotton fiber quality. The final objective of the current project was to evaluate transgenes efficacy against cotton bollworm (H. armigera) and whitefly (Bamisia tabaci). The results showed that all the transgenic cotton lines were significantly resistant to H. armigera as compared to non-transgenic control showing mortality rates between 78%-100%. Similarly, the transgenic cotton lines L3P2, L5P3, L6P3B and L6P3C showed 95%; 89%, 89%, and 72% mortality rates respectively in the whitefly bioassay. This study was unique in a sense that a combination of Bt and plant lectin genes was used to control major chewing and sucking insects to delay resistance buildup and stable insect control management.