Development of insect-resistant transgenic cotton with chimeric TVip3A* accumulating in chloroplasts

Abstract

An optimized vip3A gene, designated as vip3A* was chemically synthesized and a thi1 gene chloroplast transit peptide coding sequence was attached to its 5' end to produce the tvip3A*. vip3A* and tvip3A* genes were transformed into Gossypium hirsutum cv. Zhongmiansuo35. Of 42 independent transformants, 36 were positive for the vip3A* or tvip3A* gene. Four independent transgenic T1 lines with single-copy insertions and unchanged phenotypes (CTV1 and CTV2 for tvip3A*, and CV1 and CV2 for vip3A*) were selected by Southern blotting, and subjected to an insect bioassay and field assessment. Four homozygous T2 transgenic lines were then selected and the amount of expressed Vip3A* protein was determined by western blotting and ELISA. The protein concentrations of CTV1 and CTV2 were about three-fold higher than those of CV1 and CV2. As expected, the Vip3A* protein of CTV1 and CTV2 were transported to the chloroplasts, where they accumulated. The Vip3A* protein concentration in the chloroplasts of CTV1 and CTV2 was about 15-fold of that of CV1 and CV2. All four transgenic lines showed 100% mortality against fall armyworm (Spodoptera frugiperda) and beet armyworm (Spodoptera exigua) by insect bioassay. Moreover, CTV1 and CTV2 exhibited 100% mortality against cotton bollworm (CBW, Helicoverpa zea), whereas CV1 and CV2 showed 75.0% and 72.5% mortality against CBW, respectively. The field bioassay indicated that CTV1 and CTV2 were more resistant to CBW than CV1 and CV2. Our results suggest that the two tvip3A* transgenic lines (CTV1 and CTV2) can be used to develop insect-resistant cultivars and could be used as a resource for raising multi-toxins-expressing transgenic cotton.

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

“…Cotton has also been transformed with a synthetic vip3A gene fused to a chloroplast transit peptide coding sequence (119). The Vip3A protein accumulated in chloroplasts, and its concentration in plants was higher than that in plants transformed with just the synthetic gene.…”

Synergistic Effects of GhSOD1 and GhCAT1 Overexpression in Cotton Chloroplasts on Enhancing Tolerance to Methyl Viologen and Salt Stresses

“…The function of THI1 signal peptide in cotton is consistent with that reported in Arabidopsis [57], who demonstrated that the transit signal peptide of THI1 protein could transfer the GFP protein into chloroplasts. In addition, we previously reported that Vip3A, a Bacillus thuringiensis toxin protein, was transferred into the chloroplasts of cotton by the transit signal peptide of Arabidopsis THI1 protein [58].…”

In-Silico Determination of Insecticidal Potential of Vip3Aa-Cry1Ac Fusion Protein Against Lepidopteran Targets Using Molecular Docking

“…Vegetative insecticidal protein (Vip3Aa) from Bacillus thuringiensis (Bt) is a candidate novel toxin because of its greater toxicity and unique receptor binding sites (Tabashnik et al, 2008 ). Cry1Ac is produced during the bacterial reproductive growth phase, while Vip3Aa is secreted during the bacterial vegetative growth phase (Wu et al, 2011 ). Both are non-homologous insecticidal proteins possessing different receptors within the midgut of Lepidoptera with unique insecticidal impact (Yu et al, 1997 ; Lee et al, 2003 ).…”

“…Previously, fusion protein was used in cotton plants to increase the concentration of Vip3A protein by producing chimeric Tvip3A * protein. Transgenic plants possessing Tvip3A * genes showed an approximately 100% mortality rate in case of beet armyworm and FAW (Wu et al, 2011 ).…”