Impact of codon optimization on vip3Aa11 gene expression and insecticidal efficacy in maize

Abstract

Introduction: Codon optimization is critical for high expression of foreign genes in heterologous systems. The vip3Aa11 gene from Bacillus thuringiensis is a promising candidate for controlling Spodoptera frugiperda.

Methods and results: To develop insect-resistant maize, we designed two codon-optimized vip3Aa11 variants (vip3Aa11-m1 and vip3Aa11-m2) based on maize codon usage bias. Both recombinant proteins expressed in Escherichia coli exhibited high insecticidal activity. However, in transgenic maize, Vip3Aa11-m1 exhibited strong insecticidal activity against Spodoptera frugiperda and Spodoptera exigua, while Vip3Aa11-m2 lost activity despite identical amino acid sequences. RT-PCR analysis confirmed that both genes were transcribed correctly, but western blot results demonstrated a smaller product for vip3Aa11-m2, suggesting a translation-level alteration. Segment replacement and point mutation experiments in maize protoplasts demonstrated that the synonymous codon AAT (Asn) at the fourth amino acid position in vip3Aa11-m2 was associated with the production of a truncated protein, suggesting that the AAT codon may influence the selection of the translation initiation site, potentially shifting it to a downstream ATG (Met) codon.

Discussion: These findings not only reveal the critical role of codon context in translation initiation and protein integrity but also provide a novel strategy for optimizing foreign genes in crop improvement, particularly offering valuable insights for engineering insect-resistant maize using Bt genes.

Keywords: Spodoptera frugiperda; codon optimization; synonymous codon substitution; transgenic maize; translation initiation; vip3Aa11 gene.

Figure 1

Comparison of codon frequency; GC content; CAI; FOP and tAI among the original vip3Aa11; vip3Aa11-m1 and vip3Aa11-m2. CAI, Codon Adaptation Index; FOP, Frequency of Optimal Codons; tAI, tRNA Adaptation Index.

Figure 2

Prokaryotic expression of Vip3Aa11-m1 and Vip3Aa11-m2 proteins and analysis of their insecticidal activity against fall armyworm (FAW). (a) Schematic diagram of the prokaryotic expression vectors. (b) SDS-PAGE analysis of Vip3Aa protein expression before and after IPTG induction. M: PageRuler™ prestained protein ladder (10-180 kDa); V: commercial Vip3Aa protein (AA1611); SN: supernatant; P: pellet. The black triangle indicates the position of the Vip3Aa fusion protein. (c) Western blot analysis of the purified Vip3Aa11-m1, Vip3Aa11-m2, Vip3Aa19 and Vip3Aa20 proteins. The commercial Vip3Aa protein (V) was used as a positive control. M: PageRuler™ prestained protein ladder (10-180 kDa). (d) Leaf dip bioassay of the insecticidal activity of E.coli-expressed Vip3Aa proteins against FAW. The leaf disks dipped in PBS buffer containing 0.1% (v/v) Triton X-100 served as negative control. Data represent means ± SD (n=3 biological replicates).

Figure 3

PCR screening of the regenerated maize plants and Enzyme-linked immunosorbent assay (ELISA) of Vip3Aa11 expression in transgenic maize plants. (a) Schematic diagram of the maize genetic transformation constructs pVP1 and pVP2. The glyphosate resistance gene cp4-epsps and glufosinate resistance gene bar were used as selected marker genes for maize transformation, respectively. (b) PCR analysis of the vip3Aa11-m1 and vip3Aa11-m2 genes in corresponding transgenic maize plants. M: 5kb Plus DNA Marker (100-5000 bp); P: positive control pVP1 or pVP2 vector; B: blank; N: wild-type B104 plants. (c) Detection of Vip3Aa11 protein expression in transgenic maize plants by ELISA. **P<0.01, determined using Welch’s t-test.

Figure 4

Laboratory bioassays of VP1 and VP2 transgenic maize plants with fall armyworm (FAW) and beet armyworm (BAW). (a) The appearance of wild-type B104 and transgenic maize leaves after insect bioassays with FAW. Photographs were taken after 5 days of infestation. Scale bar=1 cm. (b) The appearance of wild-type B104 and transgenic maize leaves after insect bioassays with BAW. Photographs were taken after 3 days of infestation. (c) The mortality rates of BAW larvae feeding on the leaves of wild-type B104 and VP1-1, VP2-113, VP2-126, VP2-142 transgenic maize plants. Data represent means ± SD (n=5 biological replicates). **P<0.01, determined using one-way ANOVA.

Figure 5

PCR, RT-PCR, and immunoblot analysis of the size of vip3Aa11-m2 gene and Vip3Aa11-m2 protein in VP2 transgenic maize plants. (a) The full-length vip3Aa11-m1 and vip3Aa11-m2 genes were cloned from the genomic DNA and cDNA of VP1 and VP2 transgenic maize plants using PCR and RT-PCR, respectively. M: 5kb Plus DNA Marker (100-5000 bp); P: positive control pVP2 vector; B: blank; N: wild-type B104 plants. (b) Immunoblot analysis of the sizes of Vip3Aa11-m1 and Vip3Aa11-m2 proteins in VP1 and VP2 transgenic maize plants. M: prestained protein ladder (10-180 kDa). P: commercial Vip3Aa protein.

Figure 6

Immunoblot analysis of the size of recombinant Vip3Aa11 protein expressed in maize protoplasts. (a) Schematic diagram of the protoplasts transformation vectors. The vip3Aa11 gene was divided into three segments: A (1-789), B (790-1578), and C (1579-2370). The different segments were substituted with those of vip3Aa11-m1 and vip3Aa11-m2 gene, resulting in six recombinant vip3Aa11 gene expression vectors. (b) The ratio of normal size (N) Vip3Aa11 protein to small size (S) Vip3Aa11 protein expressed by six recombinant vip3Aa11 genes, as shown in Figure (c) The calculation of the ratio was preformed using ImageJ 1.54g software. (c) Western blot analysis of the size of six recombinant Vip3Aa11 proteins (2A1B1C, 2A2B1C, 2A1B2C, 1A2B2C, 1A1B2C, and 1A2B1C) expressed in maize protoplasts. M: prestained protein ladder (10-180 kDa). P: Vip3Aa11-m1 protein expressed in VP1-1 transgenic maize plants was used as positive control; B: The empty vector was used as a blank control. (d) Schematic diagram of the protoplasts transformation vectors. The vip3Aa11-A segment was divided into three segments: D (1-264), E (265-528), and F (529-789). The different segments were substituted with those of vip3Aa11-m1 and vip3Aa11-m2 gene, resulting in six recombinant vip3Aa11 gene expression vectors. (e) The ratio of normal size (N) Vip3Aa11 protein to small size (S) Vip3Aa11 protein expressed by six recombinant vip3Aa11 genes, as shown in Figure (f) The calculation of the ratio was preformed using ImageJ 1.54g software. (f) Western blot analysis of the size of six recombinant Vip3Aa11 proteins (2D1E1F2G, 1D2E1F2G, 1D1E2F2G, 2D1E1F1G, 1D2E1F1G, and 1D1E2F1G) expressed in maize protoplasts. M: prestained protein ladder (10-180 kDa). P: Vip3Aa11-m1 protein expressed in VP1-1 transgenic maize plants was used as positive control; B: The empty vector was used as a blank control.

Figure 7

The codon of the N4 was crucial for the translation of the Vip3Aa11 protein. (a) Sequence alignment of the first 120 bp of the vip3Aa11-m1, vip3Aa11-m2, vip3Aa19, and vip3Aa20 genes. The sequence of vip3Aa19 and vip3Aa20 genes were derived from the DBN9501 and MIR162 events, respectively. The codons that differ between vip3Aa11-m2 and the other three genes (vip3Aa11-m1, vip3Aa19, and vip3Aa20) were highlighted in green. The codons of M34 and M36 were highlighted in blue. (b) Schematic diagram of the protoplasts transformation vectors. The first 102bp and 108bp of the vip3Aa11 gene were substituted with those of vip3Aa11-m1 and vip3Aa11-m2 genes, resulting in four recombinant vip3Aa11 gene expression vectors. (c) The ratio of normal size (N) Vip3Aa11 protein to small size (S) Vip3Aa11 protein expressed by four recombinant vip3Aa11 genes, as shown in panel (d). (d) Western blot analysis of the size of four recombinant Vip3Aa11 proteins (2H1I, 2J1K, 1H2J, and 1J2K) expressed in maize protoplasts. M: prestained protein ladder (10-180 kDa). P: Vip3Aa11-m1 protein expressed in VP1-1 transgenic maize plants was used as positive control; B: The empty vector was used as a blank control. (e) Schematic diagram of the protoplasts transformation vectors. The codons N2, N4, and T6 in the vip3Aa11-m2 gene were mutated to match those in the vip3Aa11-m1 gene, resulting in the construction of seven expression vectors. (f) The ratio of normal size (N) Vip3Aa11 protein to small size (S) Vip3Aa11 protein expressed by seven mutated vip3Aa11-m2 genes, as shown in panel (g). (g) Western blot analysis of the size of Vip3Aa11-m2 proteins (N2, N4, T6, N2N4, N2T6, N4T6 and N2N4T6) expressed in maize protoplasts. M: prestained protein ladder (10-180 kDa). P: Vip3Aa11-m1 protein expressed in maize protoplasts was used as positive control; B: The empty vector was used as a blank control.