Transformation and functional verification of Cry5Aa in cotton

Abstract

Most of the cotton bollworm-resistant genes applied in cotton are more than 20 years and they all belong to Cry1Ab/c family, but the insect-resistant effects of Cry5Aa on cotton were rarely reported. The possible risk of resistance is increasing. The study synthesized a novel bollworm-resistant gene Cry5Aa artificially based on preferences of cotton codon. The new gene was transferred to cotton through the method of pollen tube pathway. The transgenic strains were identified by kanamycin test in field and laboratory PCR analysis. Meanwhile, an insect resistance test was conducted by artificial bollworm feeding with transgenic leaves and GK19 was used as a control in this study. Results showed that rate of positive transgenic strains with kanamycin resistance in the first generation (T1), the second generation (T2) and the third generation (T3) respectively were 7.76%, 73.1% and 95.5%. However, PCR analysis showed that the positive strain rate in T1, T2 and T3 were 2.35%, 55.8% and 94.5%, respectively. The resistant assay of cotton bollworm showed that the mortality rate of the second, third and fourth instar larva feed by the transgenic cotton leaves, were 85.42%, 73.35% and 62.79%, respectively. There was a significant difference between transgenic plant of Cry5Aa and GK19 in insect resistance. Finally, we also conducted the further analysis of gene expression patterns, gene flow and the effect on non-target pest in the study. The results showed that Cry5Aa gene had less environmental impact, and Cry5Aa has been transferred successfully and expressed stably in cotton. Therefore, the novel bollworm resistance gene can partially replace the current insect-resistance gene of Lepidoptera insects.