Development of Insect-Resistant Transgenic Cotton with Chimeric TVip3A* Accumulating in Chloroplasts.

An optimized vip3A gene, designated as vip3A*, was chemically synthesized, and a chloroplast transit peptide sequence of thi1 gene was attached to its 5' end to produce the tvip3A*. vip3A* and tvip3A* genes were transformed into Gossypium hirsutum cv. Zhongmiansuo35 mediated by Agrobacterium tumefaciens. Four independent transgenic T1 lines with single-copy insertions and comparable phenotypes (CTV1 and CTV2 for tvip3A*, and CV1 and CV2 for vip3A*) were selected by polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blotting, enzyme-linked immunosorbent assay (ELISA), and insect bioassay. As expected, the Vip3A* protein of CTV1 and CTV2 were transported to the chloroplasts, where they accumulated. Our results suggest that the two tvip3A* transgenic lines, CTV1 and CTV2, can directly develop insect-resistant cultivars and could be used as a resource for raising multi-toxin-expressing transgenic cotton.