ACQUIREMENT OF TRANSGENIC COTTON (GOSSYPIUM HIRSUTUM L.) RESISTANT TO HERBICIDE AND INSECT USING GLYPHOSATE-TOLERANT aroAM12 GENE AS A SELECTABLE MARKER

• Main Authors: Xie Longxu, Li Fen, Chen Mingxia, Lu Longdou
• Format: Article
• Language: English
• Published: "Alexandru Ioan Cuza" University of Iași 2005-08-01
• Series: Analele Ştiinţifice Ale Universităţii Alexandru Ioan Cuza din Iași,Sectiunea II A : Genetica si Biologie Moleculara
• Online Access: http://www.gbm.bio.uaic.ro/index.php/gbm/article/view/294
• ISSN: 1582-3571; 2248-3276

A new binary vector, pAM12-S1m, harboring the aroAM12 gene encoding for 5-enolpyruvyl- shikimate- 3-phosphate synthase (EPSPS) and a synthetic recombinant BtS1m toxin gene consisting of 331 N-terminal amino acids of CryIAc and 284 C-terminal amino acids of CryIAb has been constructed. The truncated aroAM12 gene, which was obtained through gene shuffling technology, was ligated with a transit sequence of Arabidopsis EPSPS and expressed in cotton plants driven by cauliflower mosaic virus 35S (CaMV35S) promoter. The chimeric BtS1m toxin gene was fused with DNA sequence encoding PR1b secretary signal peptide and expressed under the control of 2E-35S promoter and “ȍ” translation enhancer sequence derived from tobacco mosaic virus. The mutant EPSPS of aroAM12 gene product conferring highly resistant to glyphosate, the active ingredient in herbicide Roundup ® , was used as a dominant selectable marker for cotton plant transformation. The genes were introduced into commercial cultivar Zhongmian12 of cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation. The transformants were directly selected on medium supplemented with 80μmol/L glyphosate. In this research, 40 regenerative cotton plantlets were obtained through screening. Integration of aroAM12 and Bts1m genes was confirmed by PCR and Southern blot, the results indicated that all the 40 plants possessed the aroAM12 gene, 28 of which possessed both the aroAM12 and BtS1m genes. Expression of both the genes was established by Western blots. Insect bioassay and glyphosate resistance assay indicated that the transgenic cotton plants obtained were highly resistant to glyphosate and insect. The results of glyphosate resistance and insect bioassay of T1 generation showed that the numbers of resistance and sensitive phenotypes showed Mendelian segregation ratio.