Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene

Abstract

Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.

Keywords: Bt gene stacking; Bt resistance management; EPSPS; Transgenic rice.

Fig. 1

Schematic diagram of the T-DNA vector used for rice transformation The T-DNA contained three expression cassettes: the cassette for Bt insecticidal gene Cry1Ac under control of modified cauliflower mosaic virus 35S promoter (the original 35S promoter linked with the first intron of rice Actin-1 gene, p35sM), the cassette for Bt insecticidal Cry1Ig under control of maize ubiquitin promoter (pUbi), and the cassette for glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene (G10) under control of pUbi promoter. Cry1Ac, Cry1Ig, and G10 were synthesized genes fused with the coding sequence for transit peptide derived from maize acetohydroxy acid synthase at their 5' terminus. RB and LB indicate the right border and left border of the T-DNA, respectively

Fig. 2

Southern blot analysis of the selected transgenic events Genomic DNAs prepared from GAI-10, 14, 56, and 71 plants were digested with BamHI and then hybridized with a digoxigenin (DIG)-labelled probe specific to gene G10. M: the DNA ladder

Fig. 3

Western blot analysis of transgene expression Six plants were selected. Each sample was blotted with a primary antibody against Cry1Ac (a), Cry1Ig (b), and G10 (c), respectively. Prokaryotic expressed Cry1Ac, Cry1Ig, and G10 were used as a positive control (CK⁺), respectively. The sample prepared from non-transgenic rice was used as a negative control (CK⁻)

Fig. 4

Mortalities of cotton bollworm (CB), striped stem borer (SSB), and rice leaf roller (RLR) feeding on GAI-14 Non-transgenic rice at the same growing stage was used as the control (CK). Data are expressed as mean±standard deviation (n=3). Mortalities of all insects feeding on GAI-14 plants were 100%. ^** on the bars indicated extremely significant difference between CK and GAI-14 (P<0.01, Kruskal Wallis test)

Fig. 5

Insect bioassay for GAI-14 The bioassay was conducted with cotton bollworm (a), striped stem borer (b), and rice leaf roller (c), respectively. Non-transgenic rice at the same growing stage was used as the control (CK)

Fig. 6

Glyphosate-resistant assay of GAI-14 The GAI-14 plants were sprayed with Roundup diluted at 1:140 (v/v). Non-transgenic rice at the same growing stage was used as a control (CK)